I have isolated total RNA from FFPE tissues but the concentration is very low (1-2 ng/uL), the amount of 2-5 ng in reaction would work with the analysis of gene expression using sybrgreen in qRT-PCR ?
Hi,
You need to have 1 µg of RNA for getting good concentration and proper cDNA and I suggest you to reisolate your RNA and to get the good concentration you can precipitate your samples with Yeast tRNA OR Glycogen.
Hi,
it depends on your needs and your application. In case you are only looking for one or two genes and the expresison of these genes is moderate to high you could think og doing a one-Step Sybr RT PCR. This would safe material and in priciple should work with low inputs. In general the amount of RNA needed for reverse transcription is dependent on the used kit and the application. if you need to screen many different genes it is optimal to start with higher amounts of RNA but in cases like yours smaller amounts work. I know data from people that started with one cell and conducted qPCR analysis with this. So before reextracting perhaps you should give it a try.
Good Luck
Sven
For gene expression analysis, you can use 500ng of total RNA for cDNA synthesis.
Best of Luck.
Dear Lahmadi,
you can use Superscript cDNA synthesis kit from Invitrogen, it can convert RNA to cDNA with very minimal amount of RNA (10ng) and increase the incubation time.
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