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What went wrong in my qPCR?


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27 replies to this topic

#16 shankares

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Posted 08 February 2011 - 11:59 PM

Hello everyone, I need help with my PCR. I am new to the PCR and my aim is to detect the expression of specific genes by RT-PCR. I have enough total RNA (detected by gel electrophoresis, nanodrop) but after trancription I cant detect any cDNA. I dont know where I go wrong since I add all the reagents (random primers-70 C for 10 mins followed by 5 X buffer, 0.1M DTT, dNTPs and superscript II- 42 C for 50 mins).I would be happy if someone could help or give suggestions.

#17 jonas albarnaz

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Posted 10 April 2011 - 10:39 PM

In my lab, we also had problem with contamination in real time PCR, by using SYBR Green in an ABI machine. We solved the problem by adjusting primer and dNTP concentrations in mastermix. These reagents in excess (i.e. in concentrations needed for conventional PCR) can cause inespecific reactions and contamination, since high sensitivity of real time PCR. I suggest also to review your primers; you should design a primer pair specifically for your SYBR reaction, for instance, by using the software Primer Express supplied by ABI. Primers for TaqMan PCR or conventional PCR not necessarily fit well for SYBR Green real time PCR. Good luck!

#18 sciencelover

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Posted 16 July 2011 - 07:08 PM

Are you using good sterile [url="[url]http://www.alkaliscientific.com/en/filter-pipette-tips/637-a-barrier-tips.html"]barrier[/url] pipette tips[/url] when loading your samples? This can be the source of many of your contamination issues.

#19 rfardid

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Posted 02 January 2012 - 04:03 AM

Hi

I'm working in frozen heart tissue of rat for RNA extraction. These tissue were frozen in -20 degree from 1 years ago! But I need extraction RNA from these. I tired several times by Absolutely RNA® Miniprep Kit (Stratagene) for extracting RNA. I didn't any band in electrophoresis gel, but nanodrap spectrometer was shown good ratio about 1.9 (A260/A280). so I synthesized cDNA for QPCR from that RNAs.
After QPCR I showed amplifying is good and efficiencies is good but no-RT cDNA as negative control for gDNA contamination had amplify too!
I don't know about this problem! If does gDNA exist or RNA degrade?
Thank you for your advice...

Edited by bioforum, 17 July 2012 - 06:24 PM.


#20 WSN

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Posted 06 January 2012 - 08:48 AM

You were killing RNA by 70 dC for 10 min, you can skip denaturing all together, at least denature for shorter time, say, 2 min.

#21 WSN

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Posted 06 January 2012 - 08:54 AM

Hello, rfardid, the no-RT should be at least 10 Ct behind yes-RT, otherwise you may conclude that the amplification is all from gDNA. Design a primer pair to span a splicing junction to ignore the gDNA. Pick assay from ABI with _m1 tag. Good luck!

#22 ZEYNEP

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Posted 20 February 2012 - 06:18 AM

hi everyone,

I have a problem with my ct values (target genes and the endo controls)
Let me give you an example; for the same sample 2 different experiments have been made and every sample has done in duplicates.
First problem is ct values from the duplicates of the first experiment they don't match, there are 1 2 ct sometimes 3 ct difference between the replicas.
The second problem is when they do match ( replicas of first date match with each other and so the second date's), there is difference in ct values up to again 1 2 sometimes 3 4 ct between the different date's experiment for the same sample.

We use taqman primer probes and ABI 7500 v2.0.5 for analyses.

We won't consider pipeting til we rule out other options which i have no idea of.
For the first one we may think of pipeting but as i say it ' s not option that my mentor will accept.
but the for the second one i have no idea at all.
Could you please help me ??

#23 Gile88

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Posted 21 March 2012 - 03:05 AM

Hello everyone,

I sincerely hope that someone can help me with this problem. I really don't have a clue what's going on...
I've isolated RNA from rat's brown adipose tissue using Trizol. RNA integrity and concentrations were great (on gel and also nanodrop measurements - 260/280 around 2, and 260/230 higher than 260/280 as it should be...). cDNA was made using High capacity cDNA Reverse Transcription Kit (Applied Biosystems).
Problem is:
After regular PCR reaction (40 cycles, cDNA concentration 5ng/μl) I got visible products on agarose gel after electrophoresis for all samples and genes. Then after using same cDNA concentration and even higher in Real Time PCR (Power SYBR Green PCR Master Mix-Applied Biosystems), there wasn't any signal at all... How can that be possible?
Tomorrow, I'm going to put this samples from Real Time PCR on agarose gel to see if there are any PCR products... If there are none, what could be the problem??
Please help! I would appreciate any ideas and suggestions.

#24 phage434

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Posted 21 March 2012 - 04:31 AM

You may be adding far too much of your cDNA to the PCR reaction. Try adding a much smaller amount (10x - 1000x) or dilute the cDNA prior to adding it.

#25 Gile88

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Posted 23 March 2012 - 03:31 AM

Thanks for the advice!

Here's a new thing... After loading samples from Real Time PCR reaction on agarose gel, there were clear bands (visible products of Real Time PCR reaction). So is it possible that there is no problem in enzyme reaction (polymerization), but in detecting the signal (SYBR) or something like that?

#26 Curlis

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Posted 16 May 2012 - 01:41 PM

Hi,

I am new in qRT PCR. I ran around 5 times to troubleshoot my qRT PCR result. In mixture I am using double distilled autoclaved water and using SYBR green for this.

In disassociation curve I am getting multiple peaks. I know it can be because of primer dimer. But I am getting desired length of product which is required when I ran sample on gel after qRT-PCR. It just one band I am able to see when running qRT PCR product on gel. I am using 50Microlitre of reaction mixture for one well. I am using dliuted SYBR Green (10,000X) to 1X, .01X, .05X, 0.5X. I thought might be SYBR green is an issue, therefore I diluted it more.

I am unable to figure it out, can you please help me.

#27 biznatch

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Posted 28 May 2012 - 09:30 PM

Hi,

I am new in qRT PCR. I ran around 5 times to troubleshoot my qRT PCR result. In mixture I am using double distilled autoclaved water and using SYBR green for this.

In disassociation curve I am getting multiple peaks. I know it can be because of primer dimer. But I am getting desired length of product which is required when I ran sample on gel after qRT-PCR. It just one band I am able to see when running qRT PCR product on gel. I am using 50Microlitre of reaction mixture for one well. I am using dliuted SYBR Green (10,000X) to 1X, .01X, .05X, 0.5X. I thought might be SYBR green is an issue, therefore I diluted it more.

I am unable to figure it out, can you please help me.


I've had situations like this. Sometimes you'll see just one band on the gel but get multiple peaks in the melting curve. This could be because there are actually two peaks on the gel but they are too close together (running your gel longer may help) or there could be faint secondary products that you can't see on the gel, as the realtime PCR melt curve is a lot more sensitive that an agarose gel. Unless both my gel and melt curve indicate one clear product I don't trust the results and I either optimize the primers or design new ones. You could try an annealing temperature gradient and see how that affects what the melt curve and gel look like.

Edited by biznatch, 28 May 2012 - 09:32 PM.


#28 Curlis

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Posted 29 May 2012 - 06:38 AM


Hi,

I am new in qRT PCR. I ran around 5 times to troubleshoot my qRT PCR result. In mixture I am using double distilled autoclaved water and using SYBR green for this.

In disassociation curve I am getting multiple peaks. I know it can be because of primer dimer. But I am getting desired length of product which is required when I ran sample on gel after qRT-PCR. It just one band I am able to see when running qRT PCR product on gel. I am using 50Microlitre of reaction mixture for one well. I am using dliuted SYBR Green (10,000X) to 1X, .01X, .05X, 0.5X. I thought might be SYBR green is an issue, therefore I diluted it more.

I am unable to figure it out, can you please help me.


I've had situations like this. Sometimes you'll see just one band on the gel but get multiple peaks in the melting curve. This could be because there are actually two peaks on the gel but they are too close together (running your gel longer may help) or there could be faint secondary products that you can't see on the gel, as the realtime PCR melt curve is a lot more sensitive that an agarose gel. Unless both my gel and melt curve indicate one clear product I don't trust the results and I either optimize the primers or design new ones. You could try an annealing temperature gradient and see how that affects what the melt curve and gel look like.



Hi

Thank you for your reply. I figured out the problem; it was of SYBR Green Now it is working fine. But my graph which generated from qRT PCR looks weird. I ran it on ABI PRISM 7500.
Everything is the same as above ( Master mix).

I have attached that file in the word format.

Attached Files






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