Whole lung Tissue cDNA, Conventional PCR works but nit SYBR Green

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Metin
Metin's picture
Whole lung Tissue cDNA, Conventional PCR works but nit SYBR Green

 Dear All,
I extracted mRNA from whole lung tissues and cDNAs were synthezed

I made a conventiional PCR for GAPDH and it works well but even if i am using the same primer for GAPDH sybr green real time PCR doesnt work. My previous made cell line cDNAs works but not whole lung tissues.

Do you think any specific reason why whole tissue cDNA doesnt work?
thank you

Ivan Delgado
Ivan Delgado's picture
Hi Metin,

Hi Metin,

My first observation would be that standard PCR assays do not necessarily work when you use the same primers for a SYBR Green PCR. Most qPCR assays are designed to amplify smaller amplicons, ~200 bp, so if your standard PCR amplifies a larger piece of DNA then maybe that is the reason why it is not working. My main recommendation in these cases is to design a new assay specificically for SYBR Green qPCR.

From your message it also seems that you did get this assay to work as a SYBR Green qPCR using a different cDNA (from a cell liine). If that is the case then your lung tissue cDNA may be the issue. If the lung tissue RNA was not that good quality, maybe a little degraded, the cDNA may not contain long enough pieces of DNA to amplify your GAPDH amplicon. In that case you may want to consider extracting RNA again and making a new batch of cDNA.

Metin
Metin's picture
 Thank you Ivan

 Thank you Ivan
Sounds like the cDNAs are the problem