RNA extraction from PBMC layer - how to increase RNA Concentration during RNA extraction from BLOOd (May/16/2011 )
Hi Everyone !!!
I have to extract RNA for c DNA synthesis and amplification for the detection of BCR/ABL fusion gene transcript detection. but i m not getting enough RNA concentration for cDNA synthesis i.e 1 microgram . some samples i got has good quantity of Buffy coat layer, which makesthe things easier. but in some samples even after Ficoll gradient centrifuge i m not getting enough buffy coat layer, consequently RNA yield is getting affected which is not sufficient for c DNasynthesis and consequently when i m putting up quantitative PCR, we are not getting acceptable quantity of ABL control gene...
Kindly suggest how to increase RNA concentration in this case .
Is there any way to increase buffy coat layer .
why in some samples we are getting high quantity of buffy coat layer and in some cases very low amount
how to increase RNA Conc .. in those blood samples ..
sweta rai on Mon May 16 17:25:29 2011 said:
Hi Everyone !!!
I have to extract RNA for c DNA synthesis and amplification for the detection of BCR/ABL fusion gene transcript detection. but i m not getting enough RNA concentration for cDNA synthesis i.e 1 microgram . some samples i got has good quantity of Buffy coat layer, which makesthe things easier. but in some samples even after Ficoll gradient centrifuge i m not getting enough buffy coat layer, consequently RNA yield is getting affected which is not sufficient for c DNasynthesis and consequently when i m putting up quantitative PCR, we are not getting acceptable quantity of ABL control gene...
Kindly suggest how to increase RNA concentration in this case .
Is there any way to increase buffy coat layer .
why in some samples we are getting high quantity of buffy coat layer and in some cases very low amount
how to increase RNA Conc .. in those blood samples ..
been working on blood & bone marrow samples to detect the same thing as you.
the difference between blood samples is in the WBC ...
how much elution buffer do u use ? try decreasing the amount, in the samples with low WBC.
we used to add 30ul, but in samples with low count i remember getting down till 20ul.
& it worked nicely for qPCR.
best Wishes