Internal Control - Science topic

Why are you using capillary gel electrophoresis instead of qPCR? The detection limits of capillary tubes is very limited - easy to saturate & minimal signal for detection is also high.

You can use a 1-step qPCR for your splice variants and 1-step for a housekeeping gene.

Every number reported in the financial statements originates in a system which is reliant upon internal controls. Here are a few examples which may help your literature review:

Dear Colleague,

I hope this message finds you well. Observing increased mRNA expression following CRISPR-Cas9 editing intended to create a loss-of-function mutation can be perplexing. Several factors may contribute to this unexpected result. Here, I provide a detailed and logical analysis to help you understand and address this issue:

To address the observed increase in mRNA expression, I recommend the following steps:

By systematically addressing these factors, you can better understand the underlying cause of the increased mRNA expression and take appropriate measures to achieve the intended loss-of-function outcome.

Should you have any further questions or require additional assistance, please feel free to reach out.

This list of protocols might help us better address the issue.

Thanks a lot Hanna .....I was also thinking in the same direction.....it’s not happening repeatedly .......it happening randomly.......this is primarily happening if only mastermix is placed in the tubes without any DNA and since its purely a ARMS PCR based assay with PCR clamp involving multiple sets of primers(4 to 5 different targets ) so the chance of primer dimer formation with only mastermix is very high where as in the NTC..... in place of template when we are adding Nuclease Free Water there in no amplification (it’s completely clear) as the mastermix itself is getting diluted to some extent so chance of primer dimer formation is getting reduced at the same time as specified in the kit insert (The internal control amplifies a region of exon 2 of the EGFR gene) it’s also not getting any DNA to amplify with the primer of a region of exon 2 of the EGFR gene......I would be really thankful if you kindly relate your opinion concerning the same at your convenience

Honestly, "dilute your cDNA" is a lesson it seems far too many people need to learn the hard way. For the record, even 1/20 is usually fine for qPCR (effectively this just shifts all your data 2 cycles later than 1/5 dilution, which usually is fine even for low abundance transcripts). This also makes your cDNA last a lot longer.

Glad you solved it, but be sure to tell everyone you discuss this with that "dilute your cDNA" is the way to go.

Hi Shon, you're right. The 28/18 ratio is great, but the positions seem to be off. Are you sure the lather you used was not for DNA by any chance?

Plus, may I ask what was your purification method? the product is really clean.

Yes, it is recommended to include an internal control in your TaqMan probe-based Real-time PCR assay to monitor the performance of the assay, detect potential issues, and ensure the reliability of the results. You can either design the internal control to work in a multiplex PCR with your target DNA or run separate reactions for the target DNA and the internal control.

good luck,

Djihad Chenna

Thank you so much @Emmanuel Curis

The concentration of carbon dioxide (CO2) in the atmosphere has a strong correspondence with temperature observed during the glacial cycles of the past several hundred thousand years. When the carbon dioxide concentration goes up, temperature goes up. When the carbon dioxide concentration goes down, temperature goes down. This link between global temperatures and greenhouse gas concentrations – especially CO2 – has been true throughout Earth’s history.

Lyudmil Antonov . Thank you sir. I will look into all of these.

A two company sample? Is that enough? That will not allow any generalised conclusions. You may wish to rethink your research aims and method.

Are you trying to detect a specific pathogen? If your internal control did not work, it suggests you may have a PCR inhibition - make sure you use a DNA extraction kit that removes inhibitory molecules.

As for the different results, keep in mind that EDTA blood and blood culture are very different sample types - you will have a broader range of pathogens in EDTA blood but in lower concentrations, while in blood culture you will have 1-2 pathogens (typically) at higher concentrations.

With anti-phosphoprotein-antibodies, definitely avoid milk based solutions for blocking and dilution purposes, as the phosphates in casein will catch your antibodies.

As an alternative, BSA at 1..5% m/v usually works well. If you still get no signal, replace phosphate based buffers with tris based solutions.

At this point nothing else comes to mind other than checking the calibration on your PCR machine. Did you try to run calibration standards to validate the machine?

Hi Kathrin,

we have not tried commercially available reference materials so far, however we prepared our own and used them in various methods including single EV flow cytometry (published in ) and bead-based flow cytometry ( ).

More recently we also used fluorescently tagged EVs as reference material to evaluate EV stability and storage conditions in various methods including simple bulk plate readers ( )

You don't need huge amounts for any of those methods, if you still need some of those EVs I'm happy to share those - just email me :)

Otherwise fully agree there is a high need for more rigorously characterized EV reference materials for different purposes - I know we and some other labs are working on that :)

Hope that helps,

best wishes,

Andre

Hi Hannah Ennis , now I also got the similar issue. If you have the solution, could you share with me? Thank you so much for your read.

Yes, you can carefully control the amount of RNA. I would combine this with a (synthetic) spike-in RNA to control for RT efficiency.

Yousef Shahwan

Thanks, Dr. Batra, It would be kind of help if you can share the paper link.

I could recommend the expression of genes active in colonic epithelial cells. They are frequent in stool samples, and the expression of housekeeping genes should be informative. The problem is only the number of cells in each stool sample, but I think it can be calculated as an average number of cells.

IT audit aims to assess the internal control structure's design, efficiency, effectiveness, security points, etc. while financial audit aims to assess whether the financial statements present fairly the entity's financial position, results of operations, cash flows, etc. and whether they are in accordance with the respective standards. That's why there are standards for the financial statements evaluations.

Hi, Malcolm Nobre !

Thanks for the tips! I appreciate it and will consider it carefully it all

Srinivasakumar Kp Hi, if you want to just check the viral RNA expression in sample then use N2 primer-Probe set.

In both which method you have used TaqMan method or SYBR?

Dare Ayobami below link will be help your regarding your inquiry

https://analyticalsciencejournals.onlinelibrary.wiley.com/doi/abs/10.1002/pmic.200800298?casa_token=5_60u0JuKkQAAAAA:ffBroTRFHTSwkFgTVGzj0GoMrrZdYhjIaKbMLA8FP9iBLI8N-Jt480Zl6fyFazVGC3yjP1nVuCmv1n4zlQ

Do the Internal control and Positive control primers produce any product from a regular PCR ?

Hi Erina Noé Seiler, as Salma Aktar said, you can use β-actin or GAPDH as IPC to control inhibitions. You will find PCR sets for both in the following paper: https://gene-quantification.de/overbergh-1999.pdf

I think you need to adjust your threshold and the setup of analysis. better to show the amplification plots and curves you had

@Mhd Yousuf, thanks a lot

Dear Umadevi Subramanian

These links might be useful have a look:

1. https://www.mdpi.com/2075-4418/11/6/1091/pdf

2. https://www.scielo.br/j/rimtsp/a/hbQtF546GT5MxyGTsXkBLjC/?format=pdf&lang=en

3. https://assets.publishing.service.gov.uk/government/uploads/system/uploads/attachment_data/file/926410/Understanding_Cycle_Threshold__Ct__in_SARS-CoV-2_RT-PCR_.pdf

4. https://virologyj.biomedcentral.com/articles/10.1186/s12985-021-01559-3

5. https://www.sciencedirect.com/science/article/abs/pii/S0379073821000955?dgcid=rss_sd_all

Kind Regards

I agree with the answer of Jerome Grimes

Dear Tharaka،

An endogenous/ internal controls are usually included in the assay to correct for sample to sample variations in RT-PCR efficiency and errors in sample quantification. So difinetly you need to have a houskeeping or a reference Gene in your PCR assay

...hard to say. To exclude problems with the probe (what is not likely in my opinion) you can run an agarose gel as suggested by Giorgia. I would also recommend to repeat this sample and the next day sample from the same patient. I think the most likely explanation is a very low level of the target thus the results being affected by stochastic fluctuations (Poisson distribution).

Best,

Norman

Dear Jarosław Grządziel ,

Find some detail in the following 2 articles:

Wild Guess: Maybe because a stable housekeeper may be expressed at the 34th ± 3 cycles across all the samples e.g. C and T. Therefore it doesn't matter which cycle rather a stable expression across samples is solicited.

What's the approximate size? A dimer would appear earlier in melting curves.

Tqvm for your answers Mary C R Wilson Christopher C Kelly Desalegn Abraha Gebrekidan . Am really appreciated it.

No, it is generally better have your reference reporter with a different promoter. It decreases the probability of any transcriptional cross-talk.

Also, it is better to have a low strength promoter as a reference: this way it will be the least interfering with anything in cells. And in fact, with bright luciferases like Rluc8SG or Nan-Ogluc you will have plenty of signal even with a promoter-less (!) vector.

You may need to have a stronger promoter in a reference vector, if you are using fluorescent proteins as a reference because they need relatively high protein levels to be detected in contrast to luciferases.

It depends on the pcr mix you are going to use, for example, the ideal is that the extraction control is part of the mix, containing the primers of the species you are making the qPCR. So, you would get the control curve and the target curve. An extraction control performed separately from the sample can be performed, however, the extraction can vary from that performed in the control and in the other samples, so it becomes less reliable. It is very important to have this control, because in a qPCR or rRT-PCR mix where there is no standard internal control cuvette, you can confuse negative results with poorly extracted samples, or with the presence of amplification inhibitors. Read the preprint my department has published (it has been accepted in a peer-reviewed journal and is due to be published in the next few days). He talks a little about amplification inhibitors and methodology to get around this situation in the case of rapid extraction for covid-19. Hope this helps!

Link: https://www.preprints.org/manuscript/202009.0450/v2

The hypothesis is related to the research questions. This Internal control/ fraud identification problem is related to risk management. A good risk management framework will encompass risk culture and risk controls. For fraud management it is mostly operational risk controls.

Hello.

Good luck.

Unfortunately I have no expertise in this regard.

PM

PM

This is purely due to the baseline correction error. You adjust it manually by looking at your qPCR curves, just above the noise level. You would see the Ct values for all the samples showing amplification. Also check that all samples showing amplification also shows corresponding melting curve.

this research can be uesfull

I know that our core uses a bead-beating robot for serum exosome RNA isolation. All of the salt in the serum can be tricky to allow for a good isolation. That being said the amount you get out will likely have to be analyzed by BioAnalyzer. Exogenous spike-in miRs should be used to track extraction and normalize results.

Hi there,

A lot of journals ask for antibody-independed loading controls. When you perform Ponceau S staining of the blotting membrane after transfer (you could scan/photograph it, by the way), do you see that you loaded approx. the same amount of total protein?

So you have two groups 1) Control, 2) Disease. In this you have two targets a)GAPDH and b) Gene X.

When you say your results are quite different can you expand on this? It sounds like when your GAPDH is fine then the results are fine and when the GAPDH isn't fine your gene of interest expression is low? Would that be right?

Are you using cell line or clinical material? Secondly is GAPDH the usual control gene previously cited and can Gene X effect it?

Would need some more details to really help you out for this but it is possible the regulatory genes can be down-regulated. If you can provide a bit more info that would be useful

Best

Stephen

Since the mid-19th Century, managers have used accounting information as feedback to guide their decisions -- this why Management Accounting was born.

In the 1970s, as an internal auditor for an international woodfibre company, I participated in a program where we trained an engineer from our Canadian packaging operations to review cost-saving techniques in our US operations (the least profitable but most competitive) and transfer them to our UK operations (the most profitable but lease competitive) -- the cost savings for the company was $40 million a year.

Cost savings can be everywhere in the accounting system -- it takes imagination and courage to find and implement them.

Hi all I solved the problem. It was the probes that went off. So I prepared a fresh probe stock and all went well again. Thanks.

فرضيات البحث هي التي تتحكم في ذلك

You can try oligo analyzer.

Hi!

Please check integrity of RNA, also adjust the threshold for proper baseline correction, also try at different dilutions.

Precise and productive work, and is the content of the results in the future?

Thank you Saurabh

There may be the presence of any inhibitors of amplification. I mean did you check the efficiency of your reaction? A wild guess though you may check eEF-2 or HPRT or RPS18. Nevertheless, the tool Genevestigator lets you know the best reference in your selected tissue based on many criteria like protein IHC or ICC or WB taking the data directly from protein atlas. Please try to follow MIQE guidelines if you really want meaningful data, otherwise, this whole experiment won't mean anything.

All the Best my friend from Iowa

There is reports from other labs of measuring flavones in the sigma beta-glucuronidase extract. It is an extract from Helix Pomatia. I was able to remove most of the contamination by cleaning the enzyme with an SPE column.

Yes, I did forget to add that the serum is hydrolyzed in 2 M HCl solution for 2 hrs at 90C.

I never was able to measure the apigenin in the high fat mice but we did find literature showing that apigenin decreases in obese mice and increases when the mice recover from obesity. Apparently the metabolism is higher in the HFD mice.

Run qPCR with AmelX/AmelY. This will probably give you a ratio (XY/XX).

I used qRT-PCR, in plant viruses gene expression. It is useful as a quantitative method. Best regards.

Hi Jing Jin

Use TPM to compare the relative abundances genes/transcripts. TPM is a simple fraction, where all TPMs sum to 10^6. However, FPKM, RPKM and TMM are not reliable for relative abundance since they can change across samples regardless of similar relative abundance.

Conversely, use normalized counts (including normalized FPKM, TMM and others) to compare absolute abundances. They transform the counts to a common absolute scale, making samples comparable. Never apply across sample normalisation to TPMs because they are relative values.

To summarise, TPM and normalised counts are suitable for within-sample and across-sample comparisons, respectively.

Note:

Approach relative abundances with caution as the abundance of one gene influences the relative abundances of all other genes. This is obvious for highly expressed genes, that reduce the relative abundances of all other genes regardless of their absolute abundances.

Interesting question and I follow the answers.

Hi, this is normal for a SILAC analysis in Maxquant. If you want the reverse of the given ratio e.g. L/M rather than M/L or M/H rather than H/M all you need to do is make an extra column where you calculate: 1/( M/L).

Alternatively, maxquant gives you the L, M and H intensities, so you can calculate ratios from these directly.

Best

Ed

i m working with miRNA in placental tissue as well as blood samples using TaqMan Advanced miRNA Assays,have confusion with internal control selection in advanced chemistry because U6 not reported as internal control in advanced assay.please help me for the selection of internal control

Hello Riddhi Kirit Pandya,

First of all, what do you call "controls" for RT-qPCR? Housekeeping genes ?

Indeed, results (the relative expression level of each gene) need to be normalized by two housekeeping genes (and not usually only on one); as recommended by the MIQE guidelines (Bustin et al., 2009) with the use of geometric mean to generate an accurate normalization; available at http://www.rdml.org/miqe.php.

Then, be careful to evaluate RT-qPCR results visely (fold-change, relative expression, the 2^(-ΔΔCt), etc...). After computing your RT-qPCR slopes & curves data you obtain your 2^(-ΔΔCt) data. During this procedure, you have to normalise all your data on housekeeping genes (a couple) and on a "control experiment" (associated as the "1:1" value) to compare them as ratio-values: data are presented as relative expression of your genes. --Be careful to use geometric means at this step!--

If you want more more precise and detailed informations, please see Livak and Schmittgen (2001).

There are some old discussions regarding your topic. You can see them-

Cheers!

We use this: https://www.sigmaaldrich.com/catalog/product/sigma/mp0025?lang=en&region=CA

Dear\ Aisha khodijah Kholib Jati

Hello, You can use miR-99a-5p and miR-139-5p as control in serum due to their consistency across all sample sets. Reference for this information attached.

Hope this info will help you.

Good luck

Hi Stijn Vdb ,

Thank you for your response , i was going through some of the paper and they were using Small Nuclear RNA like U5,U6 or U1 , I will try all three and see if it will work .

Thank you very much for your suggestions!

For internal control you have to use house keeping genes.

In general one of the best genes for bacteria is 16 rDNA and for Eukaryota is 18 or 23 rDNA.

In specific situation you can use other genes.

Good luck

Dear Rukiye,

It is always a little bit difficult to make PCRs with four or more primers.

(It could take a long time to establish a new multiplex PCR system!)

In every PCR system you could need a little bit different primer concentrations.

I would guess, that you take primer concentrations from this publication.

In this case you should vary your primer concentrations in your system.

Perhaps you would need higher concentrations for the primers of your control amplfikat?

Best reagrds,

Thomas