I have prepared miRNA cDNA using Taqman advanced miRNA cDNA synthesis kit and used them with Taqman qPCR Assay. Now i want to assess expression level of more miRNAs in same samples using SYBR green chemistry. so is there any chance that it can be done using SYBR green with the same cDNA??
Vikas Gaur
Yes you can.
Varkonyi-Gasic and Hellens, (2011) refer this for more info.
Dear Vikas,
You can use the same Taqman primer for SYBR green based quantification.
Dear Sankara Naynar my question is about miRNA cDNA and their use in Real time with SYBR green, not about the Taqman Primers.
Vikas Gaur Hi vikas, I would like to know if your experiment worked.
I also have a doubt in concept with your question, did you use taqman advanced assay primers to amplify with syber green or were the primers different. Thanking you in advance.
Actually my question was to perform SYBR greent based q-RT PCR using miRNA cDNA synthesized from advanced miRNA cDNA synthesis kit provided by thermo.
hi Thulaj Meharwade and Vikas Gaur
I also faced exactly the same problem a two year before what Vikas mentioned that cDNA synthesized using Thermo advanced cDNA kit can be used for SYBr gree based RT-PCR.
And I had a worst experience with Thermo advance cDNA kit, totally failed and lost 6 months to figure it out. Even I contact their technical experts with original qRT-PCR data and protocol. But they simply ignored the matter. They saying use Taqman PCR. So suggestion was invest more money. I discarded their that third class product and blasted them in emails. They even not bother to refund our money when products failed to work and then stop communication.
I shifted whole my miRNA study using Qiagen cDNA kit, miRNA primers. They are best and provide all details with the products. Their miRscript RT kit II is wonderful, there we can use total RNA isolated using Trizol and go miRNA cDNA synthesis. and that cDNA works very fine with SYBr. Today if anyone ask sure I can say Qiagen products are best for miRNA study.
Thank you Saurabh Mandal for your valuable answer. It will help lots of people.
Saurabh Mandal Thank you for the insights. I will go ahead with qiagen reagents. Vikas Gaur I might not have been clear, I actually wanted to know which primers you had used for the real time expression, did you custom synthesize them or buy it as an assay kit from thermo or qiagen.
Thulaj Meharwade As i have prepared cDNA of miRNA from this Thermo based kit (Taqman Advanced miRNA cDNA synthesis kit) therefore i used miRNA assay from Thermo for real time expression.
Hi Thulaj Meharwade
It is always good to use company designed primers for miRNA expression. I am using the Qiagen miRNA primers. In Qiagen FP & RP are pre-mixed in one vial, u just need to add water and mix. Then use 1ul directly for qRTPCR.
For miRNA primer designing, Qiagen does little chemical modification without disturbing the main universal sequence of miRNA. It is their LNA technology. It enhances stability and reduces non-specific binding.
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