I have been synthesizing cDNA using QuantiTech Rev Transcrp (Qiagen) kit to see the expression of TFF1 gene. In the label of the kit box it says that this kit synthesize cDNA which is specific for Real Time PCR. But ours is not a Real Time PCR and I could not get any result (band/expression) for a long time even though optimizing the methods many times. I checked the quality of RNA before cDNA synthesis.
Now, is it possible, this cDNA kit is not compatible for normal PCR?
Hi A.K.M,
In my experience, using a cDNA synthesis kit made for qPCR (real-time) is not recommended for RT-PCR. The cDNA synthesis kits that I have used for qPCR produce short cDNA fragments and therefore I could not amplify certain RT-PCR targets but was able to amplify others. I suggest using a cDNA synthesis kit or reverse transcriptase such as superscript III that is able to produce long cDNAs which can be used for both qPCR and RT-PCR. Also, according to the manual for your kit, the standard reverse-transcription reaction requires a 15-min incubation. In rare cases,when analyzing RNAs with a very high degree of secondary structure or if the RT-PCR product is longer than 200 bp, it may be advantageous to increase the incubation time to 30 min.
Good luck!
I don't see how this would be possible. Have you tried different primer sets? there might be an issue with the primer set themselves....does your PCR work on a DNA control? When we first started Real Time PCR we made the mistake of overloading the system...we were trying to use 5ug of total RNA, and were using the maximum amount of cDNA in our PCRs, as we expected we had a low amount of product...this lead to total failure. When we dropped the amount of RNA down to 250ug of total RNA, and we now do a minimum dilution of 1/50 of the cDNA (where we use 5ul of this in a 20ul reaction)...Now it works unbelievably well in regular PCR, or now we are doing ddPCR.
If you continue to have issues I would definitely contact tech support at Qiagen, they might be able to help you sort it out
Hi Ron, Thank you very much for you reply. Yes i changed the primer and none is working. I used 1ug of total RNA to synthesis cDNA. and used 1ug, 800 ng, 500ng, 300 ng, 200 ng of cDNA to run PCR..
I have written to Qiagen tech support couple of weeks ago from their website but yet to get their response.
Dear AKM
I would personally recommend Roche superscript. It is by far the best product for cDNA synthesis and you will get a result every time i believe.
BW
Nikos
Hi A.K.M,
In my experience, using a cDNA synthesis kit made for qPCR (real-time) is not recommended for RT-PCR. The cDNA synthesis kits that I have used for qPCR produce short cDNA fragments and therefore I could not amplify certain RT-PCR targets but was able to amplify others. I suggest using a cDNA synthesis kit or reverse transcriptase such as superscript III that is able to produce long cDNAs which can be used for both qPCR and RT-PCR. Also, according to the manual for your kit, the standard reverse-transcription reaction requires a 15-min incubation. In rare cases,when analyzing RNAs with a very high degree of secondary structure or if the RT-PCR product is longer than 200 bp, it may be advantageous to increase the incubation time to 30 min.
Good luck!
Hi AKM,
First I would figure out where your reaction is failing, is it in the RT reaction or the PCR? Are you able to amplify a housekeeping gene? If the answer is yes, than your issue is with the PCR. To troubleshoot I would start by looking at your annealing temperatures in the PCR reaction. When I start an optimization I usually running a temperature gradient on the thermocycler to determine optimal annealing temperature. I usually start with 100ng of cDNA as a template (assuming a 1:1 amplification efficiency), and start increasing the input if the amplified band is weak, but that is after I optimized the annealing temperature. I usually do this with a postive control for the RT reaction, something I know contains a high amount of my target. If I cannot amplify the positive control sample, and the housekeeping gene is amplifying fine, then I would look at redesigning the primers.
If your housekeeping gene is not amplifying, then the issue is either your RT reaction, a nuclease, an loss in polymerase activity, or an inhibitor in the PCR system (sometimes dilutions of your cDNA reaction can over come this, as Ron suggested, but it will depend on what exactly is in the RT reaction mix, which the company does not describe). I would try a serial dilution, before purchasing a different cDNA synthesis kit. For possible nucleases, clean everything (pipettes too) with a nuclease remover and use an new box of filter tips. To test your enzyme, you can try amplifying a target in genomic DNA.
If you suspect the problem is with your cDNA reaction, one thing you may consider is trying a kit that gives you more control over your reaction (ie one that comes with random hexamers AND poly T primers, and you can choose which primer set to use). I use superscript III from Invitrogen with random hexamers. This kit incorporates an RNA denaturing, and primer annealing step that ensures more efficient amplification of your target. Secondary RNA structures may result in insufficient cDNA reaction, which may explain your issue.
I hope this gives you some new ideas as to what may be preventing your amplification. Good luck!
Cassandra
HI A.K.M.
Jonny Sena above has hit the proverbial nail on the head! The kit you are using is designed specifically to amplify very short pieces of RNA target...I didn't really consider the fact that you might have been trying to amplify a larger target. Switch to superscript III and I think you will find your PCR works just fine. This enzyme has the added benefit of being able to use at higher temperatures as well...just in case it is secondary structure giving you grief.
Good Luck
Ron
Hi Jonny and Cassandra,
Thank you very much for your excellent ideas.
To clarify Cassandra,
yes I used housekeeping gene and at the very first time it worked, but the following times no result!!! I have been doing gradient temperature as well and used different concentration of cDNA. I have used 2 master mix for PCR reaction 1. My Taq Red from Bioline and the other is Top Taq Master mix from Qiagen.
I think Jonny made a very good and valid point regarding the cDNA kit. let me try with superscript III then see what happens.
All the best to you all, you have been great...
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