ChapterPDF Available

Primer design using Primer Express® for SYBR Green-based quantitative PCR

February 2015
Methods in molecular biology (Clifton, N.J.) 1275:153-164

DOI:10.1007/978-1-4939-2365-6_11

In book: Methods in Molecular Biology (pp.153-164)
Edition: 1275
Publisher: Springer Science+Business Media New York

Authors:

Amarjeet Singh

Girdhar K Pandey

University of Delhi

To quantitate the gene expression, real-time RT-PCR or quantitative PCR (qPCR) is one of the most sensitive, reliable, and commonly used methods in molecular biology. The reliability and success of a real-time PCR assay depend on the optimal experiment design. Primers are the most important constituents of real-time PCR experiments such as in SYBR Green-based detection assays. Designing of an appropriate and specific primer pair is extremely crucial for correct estimation of transcript abundance of any gene in a given sample. Here, we are presenting a quick, easy, and reliable method for designing target-specific primers using Primer Express® software for real-time PCR (qPCR) experiments.

A snapshot of Primer Express ® 2.0 showing the starting screen where primer designing process starts.

…

Snapshots from the computer screens depicting the step to analyze different vital parameters of a primer. ( a ) In Primer Express ® 2.0, details of different parameters can be viewed by clicking on the test primer

…

A Primer Express ® 2.0 screenshot showing different options for manual modifi cation of various impor-

…

Depiction of nonspecifi c binding of the primers to any unwanted sequence. Primer may bind to a nonspecifi c sequence either completely with 100 % coverage or lesser. When it is less than 100 % coverage, two main possibilities could exist, and they are shown here. ( a ) Primer may bind to the sequence from 5 ′ end but remain unzipped at 3 ′ end of nonspecifi c sequence. ( b ) Primer may not have the complementary bases (2–3 bases) at the 5 ′ end but might fi nd the complementary bases at 3 ′ end, then it will zip at 3 ′ end and amplifi cation of nonspecifi c sequence will be propagated

…

Figures - uploaded by Amarjeet Singh

Content may be subject to copyright.

Content uploaded by Amarjeet Singh

Content may be subject to copyright.

153

Chhandak Basu (ed.), PCR Primer Design, Methods in Molecular Biology, vol. 1275,

DOI 10.1007/978-1-4939-2365-6_11, © Springer Science+Business Media New York 2015

Chapter 11

Primer Design Using Primer Express

® for SYBR

Green- Based Quantitative PCR

Amarjeet Singh and Girdhar K. Pandey

Abstract

To quantitate the gene expression, real-time RT-PCR or quantitative PCR (qPCR) is one of the most

sensitive, reliable, and commonly used methods in molecular biology. The reliability and success of a real-

time PCR assay depend on the optimal experiment design. Primers are the most important constituents of

real-time PCR experiments such as in SYBR Green-based detection assays. Designing of an appropriate

and speciﬁ c primer pair is extremely crucial for correct estimation of transcript abundance of any gene in

a given sample. Here, we are presenting a quick, easy, and reliable method for designing target-speciﬁ c

primers using Primer Express

® software for real-time PCR (qPCR) experiments.

Key words Real-time PCR , SYBR Green , Primer , Expression , Primer Express

1 Introduction

After the emergence of PCR technique in early 1980s to amplify

the DNA molecules, it has been extensively used and modiﬁ ed into

several different forms to solve the problems in molecular biology.

One of the important facets of PCR-based technique is utilized in

assessing the gene expression by qualitative or quantitative mea-

sure of mRNA in the cell. Real-time quantitative RT-PCR (qPCR)

has emerged as a powerful technique to estimate the relative quan-

titative differences in the transcript level of various samples. This

technique is favored over other methods such as northern blotting,

ribonuclease protection assays, and semi-quantitative RT-PCR for

transcript analysis because lesser amount of RNA, highly reliable

quantitative assessment, and lesser efforts to generate signiﬁ cant

data in a relatively short period of time [

1 , 2 ]. Moreover, its ease of

use and high sensitivity make it a desirable method for various

applications in molecular biology and diagnostics [

3 – 6 ]. Numerous

laboratories, which perform functional genomics studies, utilize

154

qPCR method to validate tremendous amount of transcriptomic

data generated through high-throughput techniques such as gene

chip microarrays [

7 – 14 ]. Also, during the time of publication, it is

highly recommended to validate the expression data by qPCR

analysis. Obtaining high-quality and accurate expression data

depends on the design of the qPCR experiment. Like a conven-

tional PCR reaction, qPCR reaction also consists of buffer, dNTPs,

DNA polymerase, primers, and DNA template. The success of

qPCR reaction depends on the selection and use of the best pos-

sible combinations of all these components. For the transcript or

expression analysis the RNA template is transcribed into cDNA. For

speciﬁ c ampliﬁ cation, DNA and RNA templates should be rela-

tively pure of contaminating proteins and carbohydrates, and their

purity can be ascertained by measuring the OD

260 /OD

280 and

260 /OD

230 ratio of the sample using a spectrophotometer.

Uncontaminated DNA and RNA template generally have OD

260 /

280 ratios of 1.8–2.0 and OD

260 /OD

230 ratio in the range 2.0–

2.3. Genomic DNA contamination should also be removed from

RNA samples by using a DNAse enzyme and RNA samples should

not be degraded. The RNA integrity should be analyzed on a gel

or on a bioanalyzer chip. The ratio of 28S to 18S rRNA should be

approximately 2.0 in an intact RNA sample. Primers are the most

essential and important components of a PCR reaction as they

determine the desired speciﬁ c region on the template and bind

there to initiate the polymerization of a DNA sequence. SYBR

Green-based detection method is more suitable and feasible for

expression analysis of multiple genes together. SYBR Green chem-

istry is appreciated as the simplest and cheapest chemistry for real-

time PCR applications. This dye binds to the minor groove of

double-stranded DNA and ﬂ uoresces thousands times brighter

when bound than in unbound state. This implies that SYBR Green

signal increases with the progress in PCR reaction with formation

of more double-stranded DNA product. However, one major

drawback with the SYBR Green chemistry is its nonspeciﬁ c bind-

ing to any kind of double-stranded DNA and generation of non-

speciﬁ c ﬂ uorescent signal. With the detection chemistry as that of

SYBR Green, designing of proper and speciﬁ c primers becomes

more important because nonspeciﬁ c intercalation of dye within

primer dimers may produce nonspeciﬁ c ﬂ uorescence and lead to

false positive results. Here, we present an easy and reliable method

to design speciﬁ c primers for real-time qPCR experiment using

Primer Express

® software. We also discuss about consideration of

various factors such as range of GC content, melting temperatures,

length of primer, amplicon size, primer dimer formation, stem–

loop structure generation, for reliable and speciﬁ c primer design

using computational tools.

Amarjeet Singh and Girdhar K. Pandey

155

2 Materials

1. Computer with Internet : To begin the process of primer design,

a computer system with Internet accessibility is must, as initial

reference sequences will be searched, analyzed and down-

loaded from online available public databases.

2. Primer Express

software : Primer Express

® is a program from

Applied Biosystems and used to design the primers for real-

time PCR for SYBR Green

® -based assays. This software does

not require any speciﬁ c computer program to run and can be

easily installed with a normal conﬁ guration with any version of

Windows and Macintosh.

3. Reference nucleotide sequence : To design real-time PCR prim-

ers, a nucleotide sequence is prerequisite to target a speciﬁ c

region for ampliﬁ cation and select suitable primer pair.

Generally, a full-length cDNA sequence with 5′ and 3′ UTR

region is suitable as a reference sequence but if full-length

cDNA is not available, coding region (ORF) of a gene can also

be selected ( see Note 1 ).

4. Sequence analysis tools : Once the primer(s) is selected based on

the fulﬁ llment of primary requirement, it is important to scan

it for the speciﬁ city. Sequence alignment tool such as BLAST

and similar tools can be used to align the primer with selected

reference sequence and other similar sequence to ensure that

the primer(s) bind only to the desired region on the reference

sequence and not to any other nonspeciﬁ c sequence. BLAST

tool available with NCBI (National Centre for Biotechnology

Information) is more often used for this analysis but speciﬁ c

databases such as TAIR (The Arabidopsis Information

Resource) and RGAP (Rice Genome Annotation Project-

TIGR) can also be used for species-speciﬁ c homology search.

3 Methods

1. For the gene of interest, obtain the database accession ID by

keyword search or homology search in the public databases.

2. Search in NCBI or species-speciﬁ c database for the full-length

cDNA or ORF sequence, using identiﬁ ed accession ID. Here,

we are using a rice gene as an example for which the full-

length cDNA was extracted from KOME (Knowledge-Based

Oryza Molecular Encyclopedia) database (

http://cdna01.

dna.affrc.go.jp/cDNA )

3. After retrieval, copy and paste the sequence on a text ﬁ le

(notepad) FASTA format ( see Note 2 ). However, the sequence

ﬁ le can also be directly imported as GeneWorks, GenBank

sequence, EMBL, GCG, PHYLIP, PRIMER, and ASCII text

in Primer Express

® software.

3.1 Retrieval

of Reference

Nucleotide Sequence

Primer Design Using Primer Express® for SYBR Green-Based Quantitative PCR

156

1. Open Primer Express

® by double-clicking on the software

icon in the computer.

2. At the extreme left of the screen, select “File” then “New” in

the dropdown menu. Slide to the right and select “TaqMan

Probes and Primer Design” (Fig.

1 ).

3. A new screen will open and at the sequence tab a blank space

will appear where reference sequence should be uploaded

either by copy and paste ( see Note 1 ) or by clicking on “Import

DNA File” button and selecting the text sequence ﬁ le in

FASTA format (Fig.

2 ).

4. Go to the original window and click “Options” and then from

the dropdown menu select “Find Primers/Probes now”.

5. A complete list of 200 primer pairs in version 2.0 and 50 pairs

in version 3.0 will be generated. The entire list of primer com-

binations can either be viewed on the computer screen by

clicking on the “Primers” button on sequence tab with all the

important parameters such as start and end positions, length,

Tm, and % GC content (Fig.

3 ) or it can be saved by clicking

the on “Save List” button at bottom right of the sequence tab

screen and it will be automatically imported in text format, to

the folder which contains the input reference sequence.

6. This ﬁ le can be opened with MS excel and list of primers can

be analyzed for various important parameters at any time.

3.2 Generation

of Primers by Software

Fig. 1 A snapshot of Primer Express

® 2.0 showing the starting screen where primer designing process starts.

After selecting ‘File’ then ‘New’ through dropdown menu, slide toward ‘TaqMan

® Probe and Primer Design’

and select it to start primer designing

Amarjeet Singh and Girdhar K. Pandey

Fig. 2 A snapshot of Primer Express

® 2.0 screen showing the space where reference sequence can be

imported and the various features and tools used to obtain optimum primer(s)

Fig. 3 A snapshot of the Primer Express

® 3.0 showing the list of primer combinations obtained for one of the

rice gene after clicking the Primers/Probes tab. For each pair of primers (both forward and reverse) all the

important details such as start and end position on the target sequence, primer length, GC content, melting

temperature (Tm), and sequence can be obtained in a single ﬁ le

158

7. To view and analyze the different parameters of any selected

primer(s) in the software itself, click on that primer sequence

and a small window will open in Version 2.0 where different

parameters such as GC content, Tm, and length are men-

tioned (Fig.

4a ), while in version 3.0 go to ‘Tools’ tab and in

dropdown menu select ‘Primer Probe Test Tool’ and then a

small window will pop up, which gives these details about

both forward and reverse primers and additionally about the

secondary structures such as hairpin, self-dimers, and cross-

dimers (Fig.

4b ).

At step 4 , if suitable primers are not found then a pop-up

will appear with notice saying no acceptable primer pairs were

found (in version 3.0). This may happen when the reference

sequence contain a stretch of only one nucleotide repeats at

one or both the ends, especially sequences with high GC con-

tent such as rice nucleotide sequence and hence both the

primers does not ﬁ t into the parameters of the software, ﬁ xed

to get a compatible primer pair. In this case, it is advisable to

proceed for primer designing manually.

8. For the optimum efﬁ ciency of real-time PCR reaction,

primer(s) should fulﬁ ll the following conditions:

(a) Minimum primer length: 18 bases

(b) Amplicon size: 50–150 bases

(d) Tm: 58–60 °C

(e) At the 3′ end, last 5 nucleotides should not contain more

than 2 G + C bases ( see Notes 3–6 ).

(f) Low or no self-complementarity to avoid primer dimers

To check all these parameters in Primer Express

® use the

‘Primer Test’ tool. From the “File” menu select “New”, slide

towards right and choose “Primer Test Document”. Now select

the sequence tab and choose forward or reverse primer to test.

This information will guide and suggest the users to adjust the

default parameters.

To obtain the primer(s) with all these parameters in optimum

range, instead of using default settings, adjustment can be done in

many ﬁ lters. For the adjustments click on the ‘params’ tab next to

sequence tab (Fig.

5 ).

(a) Optimum annealing temperatures ( Tm ): Although the primers

generated with default settings will have comparable Tm,

which will work optimally with ABI real-time machine, but

this can be modiﬁ ed according to user’s requirement to work

with any real-time machine and obtain better results.

(b) GC content : Sometimes if primers are not obtained in the

desired range of GC content, the ﬁ lter can be relaxed or

constricted as per the requirement.

Amarjeet Singh and Girdhar K. Pandey

159

dard size for most ampliﬁ cation experiments, but if different

primer size is required (sometime bigger length primers are

required to increase the speciﬁ city), it can be achieved through

adjustments.

Fig. 4 Snapshots from the computer screens depicting the step to analyze different vital parameters of a

primer. ( a ) In Primer Express

® 2.0, details of different parameters can be viewed by clicking on the test primer

from the list of primers. A small window appears, which shows Tm, % GC, start site, and length of the primer.

( b ) In Primer Express

® 3.0, from the Tools tab a dropdown menu comes where Primer Probe Test Tool is

selected, after that a small window appears with all the important parameters including secondary structures

such as hairpin, self-dimers, and cross-dimers

Primer Design Using Primer Express® for SYBR Green-Based Quantitative PCR

160

(d) Amplicon size : Desired amplicon length can also be changed.

Maximum amplicon size of ~150 bp results in close to 100 %

PCR efﬁ ciency, and if this length is increased, it may lead to

decrease in PCR efﬁ ciency. Minimum length of amplicon is rarely

lowered and not recommended below 50 bp but it can also be

increased to create a larger amplicon to visualize on the gel.

After verifying all the necessary parameters for a compatible primer

pair, test the primer pairs for their sequence speciﬁ city.

1. From the list of primers, select the primer pair preferably

towards the 3′ end (especially when the cDNA is synthesized

using oligo-dT primer) as 3′ end UTR region is considered

unique to a nucleotide sequence.

2. Take the primer sequence and perform homology search in

the database preferably NCBI using BLAST tool. Here, select

the organism genome and choose BLASTN tool for nucleo-

tide alignment. Copy and paste the primer sequence in the

blank space, and after choosing highly similar sequences

(megablast) click the BLAST button.

3. Analyze the hits obtained from BLAST and make sure that the

test primer binds to the reference nucleotide (cDNA) sequence

with 100 % coverage and does not bind to cDNA sequence of

any other gene of same species with 100 % coverage. If primer

binds to any nonspeciﬁ c cDNA with less than 100 % coverage,

3.3 Analysis

for the Primer

Speciﬁ city

Fig. 5 A Primer Express ® 2.0 screenshot showing different options for manual modiﬁ cation of various impor-

tant primer parameters at the Params tab, to obtain an optimum combination. Red arrows are showing the

positions where various primer parameters such as Tm, GC content, Primer length, and amplicon size can be

modiﬁ ed according to requirement of user (color ﬁ gure online)

Amarjeet Singh and Girdhar K. Pandey

161

it can be used provided it should not zip toward 3′end of

nonspeciﬁ c target sequence (mismatch of 2–3 bases) so that

DNA polymerase halt there and does not propagate to pro-

duce nonspeciﬁ c amplicon (Fig.

6a ). On the other hand if the

primer remains unzipped at 5′ end but can bind complemen-

tarily at the 3′end of nontarget sequence, ampliﬁ cation of off

target can be propagated (Fig.

6b ).

4. Primer speciﬁ city can also be checked by dissociation curve

analysis after real-time PCR run, on the real-time PCR instru-

ment itself. Ideally, a dissociation curve should contain a single

peak, which indicates the speciﬁ c amplicon generation. If prim-

ers bind nonspeciﬁ cally then multiple peaks could be observed

on the dissociation curve. As a case study, we have performed

the dissociation curve analysis for one of the rice gene with dif-

ferent samples of cDNA. Two primer combinations were

selected for this analysis, one pair with complete speciﬁ city

(100 % coverage) to the target sequence and other pair with

70 % coverage and it also binds to the off targets. qPCR ampli-

ﬁ cation with nonspeciﬁ c primer combination showed multiple

peaks in different cDNA samples after dissociation curve analy-

sis (Fig.

7a ). While ampliﬁ cation from speciﬁ c primers showed

a single peak in all the samples (Fig.

7b ). Analysis of expression

data showed that this gene has higher expression values with

nonspeciﬁ c primer combination than speciﬁ c primers. This

higher expression is mainly due to the detection of additional

ﬂ uorescence of SYBR Green from nonspeciﬁ cally ampliﬁ ed

products and hence led to false positive results.

Fig. 6 Depiction of nonspeciﬁ c binding of the primers to any unwanted sequence.

Primer may bind to a nonspeciﬁ c sequence either completely with 100 % cover-

age or lesser. When it is less than 100 % coverage, two main possibilities could

exist, and they are shown here. ( a ) Primer may bind to the sequence from 5′ end

but remain unzipped at 3′ end of nonspeciﬁ c sequence. ( b ) Primer may not have

the complementary bases (2–3 bases) at the 5′ end but might ﬁ nd the comple-

mentary bases at 3′ end, then it will zip at 3′end and ampliﬁ cation of nonspeciﬁ c

sequence will be propagated

Primer Design Using Primer Express® for SYBR Green-Based Quantitative PCR

162

Dissociation Curve

0.10

0.08

0.06

0.04

DerivativeDerivative

0.02

0.00

-0.02

0.16

0.14

0.12

0.10

0.08

0.06

0.04

0.02

0.00

-0.02

60 65 70 75 80

Temperature (C)

85 90 95

65 70 75 80

Temperature (C)

85 90 95

Fig. 7 Dissociation curve analysis performed after qPCR reaction to show primer speciﬁ city. During qPCR

analysis as a case study in rice, ( a ) one of the genes showed multiple peaks in different cDNA samples depict-

ing nonspeciﬁ c binding to template whereas, when the primers were changed to be more speciﬁ c ( b ) then a

single speciﬁ c peak was observed in most of the samples at a particular annealing temperature

Amarjeet Singh and Girdhar K. Pandey

163

4 Notes

1. When using Primer Express

® , it is better to start primer design-

ing with about 500 bp of reference nucleotide sequence to

obtain more speciﬁ c and efﬁ cient primers.

2. When importing a reference sequence ﬁ le to Primer Express

software, make sure that the sequence is in a tab-delimited text

format and does not include any extraneous information such

as detail description of the sequence. This may lead to nonrec-

ognition of the sequence by the software.

3. Repeats, e.g., AGAGAGAG and runs of a nucleotide, e.g.,

GTTTTTTCG should be avoided in the sequence as they lead

to the mispriming on the template.

4. When searching for the primers (at step 3 in generation of

primers by software, methods), make sure that “Limit 3′G + C”

box is selected, otherwise primer generated without this limita-

tion will contain more than 2 G + C in the last 5 bases at 3′end.

5. For relative expression analysis using SYBR Green for several

genes, it is advisable to keep the length of the amplicon very

close because larger size product will produce more ﬂ uores-

cence, hence the expression values obtained will not reﬂ ect the

actual relative expression of multiple genes.

6. It is beneﬁ cial to design a primer crossing intron/exon bound-

ary, as it will lead to ampliﬁ cation of speciﬁ c cDNA sample and

not the genomic DNA ampliﬁ cation. In such cases, DNAse

treatment of RNA samples can be avoided.

Acknowledgement

Research work in GKP’s lab is partially supported by grants from

University of Delhi, Department of Biotechnology (DBT),

Department of Science and Technology (DST), and Council of

Scientiﬁ c and Industrial Research (CSIR), India. AS acknowledges

CSIR for research fellowship.

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4. Ginzinger DG (2002) Gene quantiﬁ cation

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Amarjeet Singh and Girdhar K. Pandey

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Mar 2023

Volatile organic compounds such as terpenes influence the quality parameters of grapevine through their contribution to the flavour and aroma profile of berries. Biosynthesis of volatile organic compounds in grapevine is relatively complex and controlled by multiple genes, the majority of which are unknown or uncharacterised. To identify the genomic regions that associate with modulation of these compounds in grapevine berries, volatile metabolic data generated via GC-MS from a grapevine mapping population was used to identify quantitative trait loci (QTLs). Several significant QTLs were associated with terpenes, and candidate genes were proposed for sesquiterpene and monoterpene biosynthesis. For monoterpenes, loci on chromosomes 12 and 13 were shown to be associated with geraniol and cyclic monoterpene accumulation, respectively. The locus on chromosome 12 was shown to contain a geraniol synthase gene (VvGer), while the locus on chromosome 13 contained an α-terpineol synthase gene (VvTer). Molecular and genomic investigation of VvGer and VvTer revealed that these genes were found in tandemly duplicated clusters, displaying high levels of hemizygosity. Gene copy number analysis further showed that not only did VvTer and VvGer copy numbers vary within the mapping population, but also across recently sequenced Vitis cultivars. Significantly, VvTer copy number correlated with both VvTer gene expression and cyclic monoterpene accumulation in the mapping population. A hypothesis for a hyper-functional VvTer allele linked to increased gene copy number in the mapping population is presented and can potentially lead to selection of cultivars with modulated terpene profiles. The study highlights the impact of VvTPS gene duplication and copy number variation on terpene accumulation in grapevine.

Analysis of Porcine DNA in Gelatine Products using SYBR Green Real Time Polymerase Chain Reaction for Halal Verification

Article

Dec 2022

Real-time PCR was employed in this study to determine whether gelatine items from nearby marketplaces contained porcine DNA. The mitochondrial NADH dehydrogenase subunit 5 (MT-ND5) gene from pigs (Sus scrofa) was the subject of oligonucleotide primer design; the forward and the reverse primer sequences produced 161bp amplicon. SYBR Green fluorescent dye was used to detect swine DNA in samples of 22 different types of gelatine products, 12 of which had labels identifying their sources and indicating their certification as Halal, and the remaining 10 had neither labels nor certification information. The 40 cycles of positive amplication with a Ct value of 19.81 served as the criterion for determining the specificity of the primers. In this work, the detection threshold for porcine DNA is set at 0.001 µg/ml. The efficiency of the PCR in this study is 91.72%, and the R2 is 0.9985, which indicates good efficiency. The 12 labelled products exhibit positive amplifications for porcine gelatine products while exhibiting negative amplifications for both bovine and fish gelatine products in labelled samples. Nine out of the ten samples of gelatine products that were tested had positive amplifications without source or Halal labelling.

Design and Validation of qPCR-Specific Primers for Quantification of the Marketed Terfezia claveryi and Terfezia crassiverrucosa in Soil

Article

Full-text available

Oct 2022
J. Fungi

Desert truffle crop is a pioneer in southeastern Spain, a region where native edible hypogeous fungi are adapted to the semiarid areas with low annual rainfall. Terfezia claveryi Chatin was the first species of desert truffle to be cultivated, and has been increasing in recent years as an alternative rainfed crop in the Iberian Peninsula. However, its behaviour in the field has yet not been investigated. For this purpose, specific primers were designed for the soil DNA quantification of both T. claveryi and Terfezia crassiverrucosa and a real-time qPCR protocol was developed, using the ITS rDNA region as a target. Moreover, a young desert truffle orchard was sampled for environmental validation. The results showed the highest efficiency for the TerclaF3/TerclaR1 primers pair, 89%, and the minimal fungal biomass that could be reliable detected was set at 4.23 µg mycelium/g soil. The spatial distribution of fungal biomass was heterogeneous, and there was not a direct relationship between the quantity of winter soil mycelium and the location/productivity of desert truffles. This protocol could be applied to tracking these species in soil and understand their mycelial dynamics in plantations and wild areas.

Molecular and expression analysis indicate the role of CBL interacting protein kinases (CIPKs) in abiotic stress signaling and development in chickpea

Article

Full-text available

Oct 2022

Calcineurin B-like proteins (CBL)-interacting protein kinases (CIPKs) regulate the developmental processes, hormone signal transduction and stress responses in plants. Although the genome sequence of chickpea is available, information related to the CIPK gene family is missing in this important crop plant. Here, a total of 22 CIPK genes were identified and characterized in chickpea. We found a high degree of structural and evolutionary conservation in the chickpea CIPK family. Our analysis showed that chickpea CIPKs have evolved with dicots such as Arabidopsis and soybean, and extensive gene duplication events have played an important role in the evolution and expansion of the CIPK gene family in chickpea. The three-dimensional structure of chickpea CIPKs was described by protein homology modelling. Most CIPK proteins are localized in the cytoplasm and nucleus, as predicted by subcellular localization analysis. Promoter analysis revealed various cis-regulatory elements related to plant development, hormone signaling, and abiotic stresses. RNA-seq expression analysis indicated that CIPKs are significantly expressed through a spectrum of developmental stages, tissue/organs that hinted at their important role in plant development. The qRT-PCR analysis revealed that several CaCIPK genes had specific and overlapping expressions in different abiotic stresses like drought, salt, and ABA, suggesting the important role of this gene family in abiotic stress signaling in chickpea. Thus, this study provides an avenue for detailed functional characterization of the CIPK gene family in chickpea and other legume crops.

A Rare MSH2 Variant as a Candidate Marker for Lynch Syndrome II Screening in Tunisia: A Case of Diffuse Gastric Carcinoma

Article

Full-text available

Jul 2022

Several syndromic forms of digestive cancers are known to predispose to early-onset gastric tumors such as Hereditary Diffuse Gastric Cancer (HDGC) and Lynch Syndrome (LS). LSII is an extracolonic cancer syndrome characterized by a tumor spectrum including gastric cancer (GC). In the current work, our main aim was to identify the mutational spectrum underlying the genetic predisposition to diffuse gastric tumors occurring in a Tunisian family suspected of both HDGC and LS II syndromes. We selected the index case “JI-021”, which was a woman diagnosed with a Diffuse Gastric Carcinoma and fulfilling the international guidelines for both HDGC and LSII syndromes. For DNA repair, a custom panel targeting 87 candidate genes recovering the four DNA repair pathways was used. Structural bioinformatics analysis was conducted to predict the effect of the revealed variants on the functional properties of the proteins. DNA repair genes panel screening identified two variants: a rare MSH2 c.728G>A classified as a variant with uncertain significance (VUS) and a novel FANCD2 variant c.1879G>T. The structural prediction model of the MSH2 variant and electrostatic potential calculation showed for the first time that MSH2 c.728G>A is likely pathogenic and is involved in the MSH2-MLH1 complex stability. It appears to affect the MSH2-MLH1 complex as well as DNA-complex stability. The c.1879G>T FANCD2 variant was predicted to destabilize the protein structure. Our results showed that the MSH2 p.R243Q variant is likely pathogenic and is involved in the MSH2-MLH1 complex stability, and molecular modeling analysis highlights a putative impact on the binding with MLH1 by disrupting the electrostatic potential, suggesting the revision of its status from VUS to likely pathogenic. This variant seems to be a shared variant in the Mediterranean region. These findings emphasize the importance of testing DNA repair genes for patients diagnosed with diffuse GC with suspicion of LSII and colorectal cancer allowing better clinical surveillance for more personalized medicine.

Morphophysiological and transcriptome analysis reveal that reprogramming of metabolism, phytohormones and root development pathways governs the potassium (K) deficiency response in two contrasting chickpea cultivars

Article

Full-text available

Jan 2023

Potassium (K⁺) is an essential macronutrient for plant growth and development. K⁺ deficiency hampers important plant processes, such as enzyme activation, protein synthesis, photosynthesis and stomata movement. Molecular mechanism of K⁺ deficiency tolerance has been partly understood in model plants Arabidopsis, but its knowledge in legume crop chickpea is missing. Here, morphophysiological analysis revealed that among five high yielding desi chickpea cultivars, PUSA362 shows stunted plant growth, reduced primary root growth and low K⁺ content under K⁺ deficiency. In contrast, PUSA372 had negligible effect on these parameters suggesting that PUSA362 is K⁺ deficiency sensitive and PUSA372 is a K⁺ deficiency tolerant chickpea cultivar. RNA-seq based transcriptome analysis under K⁺ deficiency revealed a total of 820 differential expressed genes (DEG’s) in PUSA362 and 682 DEGs in PUSA372. These DEGs belongs to different functional categories, such as plant metabolism, signal transduction components, transcription factors, ion/nutrient transporters, phytohormone biosynthesis and signalling, and root growth and development. RNA-seq expression of randomly selected 16 DEGs was validated by RT-qPCR. Out of 16 genes, 13 showed expression pattern similar to RNA-seq expression, that verified the RNA-seq expression data. Total 258 and 159 genes were exclusively up-regulated, and 386 and 347 genes were down-regulated, respectively in PUSA362 and PUSA372. 14 DEGs showed contrasting expression pattern as they were up-regulated in PUSA362 and down-regulated in PUSA372. These include somatic embryogenesis receptor-like kinase 1, thaumatin-like protein, ferric reduction oxidase 2 and transcription factor bHLH93. Nine genes which were down-regulated in PUSA362 found to be up-regulated in PUSA372, including glutathione S-transferase like, putative calmodulin-like 19, high affinity nitrate transporter 2.4 and ERF17-like protein. Some important carbohydrate metabolism related genes, like fructose-1,6-bisphosphatase and sucrose synthase, and root growth related Expansin gene were exclusively down-regulated, while an ethylene biosynthesis gene 1-aminocyclopropane-1-carboxylate oxidase 1 (ACO1) was up-regulated in PUSA362. Interplay of these and several other genes related to hormones (auxin, cytokinin, GA etc.), signal transduction components (like CBLs and CIPKs), ion transporters and transcription factors might underlie the contrasting response of two chickpea cultivars to K⁺ deficiency. In future, some of these key genes will be utilized in genetic engineering and breeding programs for developing chickpea cultivars with improved K⁺ use efficiency (KUE) and K⁺ deficiency tolerance traits.

CBL-interacting protein kinases (CIPKs) in Chickpea: Genome- wide identification, structure and expression analysis under abiotic stresses and development

Preprint

Full-text available

Jun 2022

Calcineurin B-like proteins (CBL)-interacting protein kinases (CIPKs) regulate the developmental processes, hormone signal transduction and stress responses in plants. Although the genome sequence of chickpea is available, information related to the CIPK gene family is missing in this important crop plant. Here, a total of 22 CIPK genes were identified and characterized in chickpea. We found a high degree of structural and evolutionary conservation in the chickpea CIPK family. Our analysis showed that chickpea CIPK s have evolved with dicots from common ancestors, and extensive gene duplication events have played an important role in the evolution and expansion of the CIPK gene family in chickpea. The three-dimensional structure of chickpea CIPKs was described by protein homology modelling. Most CIPK proteins are localized in the cytoplasm and nucleus, as predicted by in-silico subcellular localization. Promoter analysis revealed various cis-regulatory elements related to plant development, hormone signaling, and abiotic stresses. RNA-seq expression analysis indicated that CIPK s are significantly expressed through a spectrum of developmental stages, tissue/organs that hinted at their important role in plant development. The qRT-PCR analysis revealed that several CaCIPK genes had specific and overlapping expressions in different abiotic stresses like drought, salt, and ABA, suggesting the important role of this gene family in abiotic stress signaling in chickpea. Thus, this study provides an avenue for detailed functional characterization of the CIPK gene family in chickpea and other legume crops.

Genome-wide expressional and functional analysis of calcium transport elements during abiotic stress and development in rice

Article

Full-text available

Feb 2014
FEBS J

Ca(2+) homeostasis is required to maintain a delicate balance of cytosolic Ca(2+) during normal and adverse growth conditions. Various Ca(2+) transporters actively participate to maintain this delicate balance especially during abiotic stresses and developmental events in plants. In this study, we are presenting a genome wide account, detail expression profiles, sub-cellular localization and functional analysis of rice calcium transport elements. Exhaustive in-silico data mining and analysis resulted in the identification of 81 Ca(2+) transport element genes, which belongs to various groups such as Ca(2+) -ATPases (pumps), exchangers, channels, glutamate receptor homologs (GLRs) and annexins. Phylogenetic analysis revealed that different Ca(2+) transporters are evolutionary conserved across different plant species. Comprehensive expression analysis by gene chip microarray and qPCR revealed that a substantial proportion of Ca(2+) transporter genes were expressed differentially under abiotic stresses (salt, cold and drought) and reproductive developmental stages (panicle and seed) in rice. These findings suggest possible role of rice Ca(2+) transporters in abiotic stress and development triggered signaling pathways. Subcellular localization of Ca(2+) transporters from different groups in Nicotiana benthamiana revealed their variable localization to different compartments, which could be their possible sites of action. Complementation of Ca(2+) transport activity of K616 yeast mutant by Ca(2+) -ATPase, OsACA7 and involvement in salt tolerance verified its functional behavior. This study will encourage detailed characterization of potential candidate Ca(2+) transporters for their functional role in-planta. This article is protected by copyright. All rights reserved.

Comprehensive Genomic Analysis and Expression Profiling of Phospholipase C Gene Family during Abiotic Stresses and Development in Rice

Article

Full-text available

May 2013
PLOS ONE

Background Phospholipase C (PLC) is one of the major lipid hydrolysing enzymes, implicated in lipid mediated signaling. PLCs have been found to play a significant role in abiotic stress triggered signaling and developmental processes in various plant species. Genome wide identification and expression analysis have been carried out for this gene family in Arabidopsis, yet not much has been accomplished in crop plant rice. Methodology/Principal Findings An exhaustive in-silico exploration of rice genome using various online databases and tools resulted in the identification of nine PLC encoding genes. Based on sequence, motif and phylogenetic analysis rice PLC gene family could be divided into phosphatidylinositol-specific PLCs (PI-PLCs) and phosphatidylcholine- PLCs (PC-PLC or NPC) classes with four and five members, respectively. A comparative analysis revealed that PLCs are conserved in Arabidopsis (dicots) and rice (monocot) at gene structure and protein level but they might have evolved through a separate evolutionary path. Transcript profiling using gene chip microarray and quantitative RT-PCR showed that most of the PLC members expressed significantly and differentially under abiotic stresses (salt, cold and drought) and during various developmental stages with condition/stage specific and overlapping expression. This finding suggested an important role of different rice PLC members in abiotic stress triggered signaling and plant development, which was also supported by the presence of relevant cis-regulatory elements in their promoters. Sub-cellular localization of few selected PLC members in Nicotiana benthamiana and onion epidermal cells has provided a clue about their site of action and functional behaviour. Conclusion/Significance The genome wide identification, structural and expression analysis and knowledge of sub-cellular localization of PLC gene family envisage the functional characterization of these genes in crop plants in near future.

Comprehensive expression analysisof rice phospholipase D gene familyduring abiotic stresses and development

Article

Full-text available

Jul 2012

Phospholipase D is one of the crucial enzymes involved in lipid mediated signaling, triggered during various developmental and physiological processes. Different members of PLD gene family have been known to be induced under different abiotic stresses and during developmental processes in various plant species. In this report, we are presenting a detailed microarray based expression analysis and expression profiles of entire set of PLD genes in rice genome, under three abiotic stresses (salt, cold and drought) and different developmental stages (3-vegetative stages and 11-reproductive stages). Seven and nine PLD genes were identified, which were expressed differentially under abiotic stresses and during reproductive developmental stages, respectively. PLD genes, which were expressed significantly under abiotic stresses exhibited an overlapping expression pattern and were also differentially expressed during developmental stages. Moreover, expression pattern for a set of stress induced genes was validated by real time PCR and it supported the microarray expression data. These findings emphasize the role of PLDs in abiotic stress signaling and development in rice. In addition, expression profiling for duplicated PLD genes revealed a functional divergence between the duplicated genes and signify the role of gene duplication in the evolution of this gene family in rice. This expressional study will provide an important platform in future for the functional characterization of PLDs in crop plants.

Expression dynamics of metabolic and regulatory component across stages of panicle and seed development in indica rice

Article

Full-text available

Mar 2012
FUNCT INTEGR GENOMIC

Carefully analyzed expression profiles can serve as a valuable reference for deciphering gene functions. We exploited the potential of whole genome microarrays to measure the spatial and temporal expression profiles of rice genes in 19 stages of vegetative and reproductive development. We could verify expression of 22,980 genes in at least one of the tissues. Differential expression analysis with respect to five vegetative tissues and preceding stages of development revealed reproductive stage-preferential/-specific genes. By using subtractive logic, we identified 354 and 456 genes expressing specifically during panicle and seed development, respectively. The metabolic/hormonal pathways and transcription factor families playing key role in reproductive development were elucidated after overlaying the expression data on the public databases and manually curated list of transcription factors, respectively. During floral meristem differentiation (P1) and male meiosis (P3), the genes involved in jasmonic acid and phenylpropanoid biosynthesis were significantly upregulated. P6 stage of panicle, containing mature gametophytes, exhibited enrichment of transcripts involved in homogalacturonon degradation. Genes regulating auxin biosynthesis were induced during early seed development. We validated the stage-specificity of regulatory regions of three panicle-specific genes, OsAGO3, OsSub42, and RTS, and an early seed-specific gene, XYH, in transgenic rice. The data generated here provides a snapshot of the underlying complexity of the gene networks regulating rice reproductive development.

Rice Phospholipase A Superfamily: Organization, Phylogenetic and Expression Analysis during Abiotic Stresses and Development

Article

Full-text available

Feb 2012
PLOS ONE

Abstract BACKGROUND: Phospholipase A (PLA) is an important group of enzymes responsible for phospholipid hydrolysis in lipid signaling. PLAs have been implicated in abiotic stress signaling and developmental events in various plants species. Genome-wide analysis of PLA superfamily has been carried out in dicot plant Arabidopsis. A comprehensive genome-wide analysis of PLAs has not been presented yet in crop plant rice. METHODOLOGY/PRINCIPAL FINDINGS: A comprehensive bioinformatics analysis identified a total of 31 PLA encoding genes in the rice genome, which are divided into three classes; phospholipase A(1) (PLA(1)), patatin like phospholipases (pPLA) and low molecular weight secretory phospholipase A(2) (sPLA(2)) based on their sequences and phylogeny. A subset of 10 rice PLAs exhibited chromosomal duplication, emphasizing the role of duplication in the expansion of this gene family in rice. Microarray expression profiling revealed a number of PLA members expressing differentially and significantly under abiotic stresses and reproductive development. Comparative expression analysis with Arabidopsis PLAs revealed a high degree of functional conservation between the orthologs in two plant species, which also indicated the vital role of PLAs in stress signaling and plant development across different plant species. Moreover, sub-cellular localization of a few candidates suggests their differential localization and functional role in the lipid signaling. CONCLUSION/SIGNIFICANCE: The comprehensive analysis and expression profiling would provide a critical platform for the functional characterization of the candidate PLA genes in crop plants

Molecular Cloning: A Laboratory Manual

Article

Jan 2001

D.W.R. Joseph Sambrook

Gene quantification using real-time quantitative PCR

Article

Jun 2002
EXP HEMATOL

David Ginzinger

The recent flood of reports using real-time Q-PCR testifies to the transformation of this technology from an experimental tool into the scientific mainstream. Many of the applications of real-time Q-PCR include measuring mRNA expression levels, DNA copy number, transgene copy number and expression analysis, allelic discrimination, and measuring viral titers. The range of applications of real-time Q-PCR is immense and has been fueled in part by the proliferation of lower-cost instrumentation and reagents. Successful application of real-time Q-PCR is not trivial. However, this review will help guide the reader through the variables that can limit the usefulness of this technology. Careful consideration of the assay design, template preparation, and analytical methods are essential for accurate gene quantification.

Comparative transcriptome analysis of cadmium responses in Solanum nigrum and Solanum torvum

Article

Jul 2012
NEW PHYTOL

• Solanum nigrum is a cadmium (Cd) accumulator, whereas Solanum torvum is a low Cd-accumulating plant. The molecular mechanisms that are responsible for differential cadmium (Cd) accumulation in the two Solanum species are poorly understood. • Here, grafting experiments confirmed that increased Cd loading into the root xylem was responsible for the differential Cd accumulation in the two Solanum species. An iron (Fe) supply assay indicated that low Fe accumulation in S. torvum leaves is related to its Cd sensitivity. • Transcriptome analyses revealed higher expression of the genes that encode several metal transporters as well as antioxidant-related genes, and several organic and amino acid biosynthesis/metabolism-related genes in Cd-treated S. nigrum. Our data also indicated that the different responsive mechanisms of the transporter genes to Fe deficiency might be responsible for differential uptake and redistribution of metals in the two Solanum species • These results form a basis upon which to further explore the molecular mechanisms of Cd accumulation and tolerance, and provide an insight into novel strategies that can be used for phytoremediation and food safety.

Molecular Cloning: A Laboratory Manual (3-Volume Set)

Book

Jan 2001

Transcriptome analysis reveals coordinated spatiotemporal regulation of hemoglobin and nitrate reductase in response to nitrate in maize roots

Article

Jul 2011
NEW PHYTOL

Given the importance of nitrogen for plant growth and the environmental costs of intense fertilization, an understanding of the molecular mechanisms underlying the root adaptation to nitrogen fluctuations is a primary goal for the development of biotechnological tools for sustainable agriculture. This research aimed to identify the molecular factors involved in the response of maize roots to nitrate. cDNA-amplified fragment length polymorphism was exploited for comprehensive transcript profiling of maize (Zea mays) seedling roots grown with varied nitrate availabilities; 336 primer combinations were tested and 661 differentially regulated transcripts were identified. The expression of selected genes was studied in depth through quantitative real-time polymerase chain reaction and in situ hybridization. Over 50% of the genes identified responded to prolonged nitrate starvation and a few were identified as putatively involved in the early nitrate signaling mechanisms. Real-time results and in situ localization analyses demonstrated co-regulated transcriptional patterns in root epidermal cells for genes putatively involved in nitric oxide synthesis/scavenging. Our findings, in addition to strengthening already known mechanisms, revealed the existence of a new complex signaling framework in which brassinosteroids (BRI1), the module MKK2-MAPK6 and the fine regulation of nitric oxide homeostasis via the co-expression of synthetic (nitrate reductase) and scavenging (hemoglobin) components may play key functions in maize responses to nitrate.

Primer design using Primer Express® for SYBR Green-based quantitative PCR

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