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Inconsistent sample quality in quantitative real-time PCR - What could be the pr

real time PCR RNA extraction cDNA synthesis SYBR green Light Cycler 480

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#1 Skl

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Posted 29 October 2012 - 08:37 AM

Hello,

I need your help with qPCR problems!
For a while now I have had problems with my real-time qPCR. The quality of my cDNA samples seems to differ so much that some samples lead to good results and some show very late or no amplifications at all.

Here are the facts:

- I am using a Spin column RNA extraction (Macherey-Nagel Kit, elution in provided RNase-free Water) to extract RNA from eukaryotic cell cultures
- gDNA is degraded on the column by DNase
- my RNA samples have 260/280 values above 2.0 ( some 260/230 values are very low but these are surprisingly not the samples with bad results)
- my RNA samples are not degraded (checked with Agilgent Bioanalyzer RNA Nano 6000 chips, RIN >9)
- all RNA samples have about the same concentrations (e.g. when extracting 9 samples all are about 180-220 ng/µL - measured in Nanophotometer)

- I am using the Revert Aid First Strand cDNA synthesis kit by Thermo Scientific (formerly Fermentas) with Oligo-dT-primers for generation of cDNA only from mRNA
- I use 1 µg of RNA for each cDNA reaction (this is whithin the recommended range of the kit)
- I take 1 µL from each cDNA sample for qPCR

- For qPCR I use the Platinum SYBR Green Mastermix by Life Technologies (Invitrogen) on the Light Cycler 480 System (Roche)
- I measure 3 housekeeping genes (Actin, Gapdh and Vezatin) and 1 target gene and prepare Mastermixes for each primer-pair
- my primers are from one exon to the next (so no amplification from gDNA possible) - the amplificate is 100 bp long
- primers are checked by regular PCR and show only one band in gel with correct size in 100 bp and melting curves in those reactions that worked are fine


Now my problems:

- Some samples show normal amplification curves for all 4 genes with Cps at about 17 for Actin
- some samples show very very late amplifictaion (e.g Cp 35 for Actin) or no amplification at all
- since the whole sample is problematic in all 4 tested genes I would think that my biological experiment is not the problem - at least the houskeeping genes should be present somehow

Since I used the same Sybr--Mastermix for all samples I conclude that the Mastermix itself and the primers are fine - the problem has to be in the steps between RNA extraction and qPCR ?!!?
I thought that maybe my cDNA concentration could be to high and therefore inhibiting the qPCR but dilutions of the cDNA templates resulted in even later amplification curves.

Please, do you have any idea or suggestion what the problem might be?
Do you know if it could be a problem with inhibitors and if so, how can I get rid of them without changing my sample concentration too much?

I am grateful for any suggestions!

Edited by Skl, 29 October 2012 - 09:04 AM.


#2 Curlis

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Posted 29 October 2012 - 08:47 AM

I had the same problem but now it works fine for me. I think you don't need to make mRNA first before making cDNA. I use my home made recipe for this. I use total RNA and convert that into cDNA. Titrate your RNA with primer and water.

Heat it at 65 degree for 10 minutes, then cool it on ice for 5-6 min, then mix your RT enzyme, buffer and Mgcl2 mix it properly and keep it at 42 degree for 1 hr.

Then do a normal PCR to confirm it. Use the same condition as PCR but this time include SYBR green.

Suggestion: run those product (which you get from qRT-PCR) on a gel, which will give you an idea about your product. Do pipetting as less as possible, because most of the time pipetting error creates problem in this experiment.

#3 Skl

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Posted 29 October 2012 - 09:01 AM

Hey ashu2007,

thanks for your reply. Sorry, I wrote that part with a misunderstanding and just edited my first post: of course I am not creating mRNA...I use the Oligo-dT-primers in the cDNA synthesis to just have cDNA from the mRNAs. Sorry for the confusion.

The procedure that you explain is essentially what is done in the cDNA synthesis kit: primers are added to RNA+water, mixture is denaturated for 5 min at 65 °C, buffer, RT enzyme, RNase inhibitor and dNTPs are added and mixture is incubated at 42 °C for 1 hr.

Yes, I can do a gel from my sample after qPCR but if I have no amplification curve I would not exprect to see a band? What should I be looking for?

#4 Trof

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Posted 04 November 2012 - 03:33 PM

If you have good quality RNA of same concentration, validated by gel or Bioanalyzer, there are only two options.
First you have serious errors in sample preparation (this could be tested running the sample multiple times more carefully) or problems with certain wells (if this is a repeated pattern in all runs) or something this kind related. You could also try normal PCR amplification (different enzyme/mix conditions) just to test it's not something in the real-time workflow.

The other is inhibitors, though it will be strange that they are only present in some samples if the isolation is identical. Test for this is diluting the template 10x, 100x or more and amplifing it. If it gets higher amplification than a 10x dilution should (compared with the good samples diluted) or even better amplification than undiluted, it's the inhibitors. Aslo adding some template like DNA to the bad samples and trying to amplify it would tell you something.

You can put the result of qPCR on gel anyway to test there really isn't any product, it would be unlikely in case of SYBR but if you have the product, why not try that. If you had for some reason problems with fluorescence imaging only, product woudl show on gel. You have the product anyway, so why not test all posibilities.

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