I have tried to isolation RNA from human dendritic cells using RNeasy kit (QIAGEN), but I got a very low yield of RNA, such 30ng/ul.
In the last time, I tried to combine QIAGEN protocol with Trizol protocol, because I had some samples (10^5 cells) incubated with Trizol at -80ºC, so I added 0,2 ml of chloroform for each 1 ml of Trizol and the samples was centrifugated at 12.000 x g for 15 min and the aqueous fase was recovered, I skiped the step that RLT buffer is added and I added 1 volume of ethanol 70% to the lysate and I proceed with the RNeasy protocol using the columns, but I got only 11ng/ul of RNA.
I need this RNA to synthesize cDNA and afterwards performe qPCR
Am I doing something wrong or the kit RNeasy is not a good way to obtain RNA? Using Trizol protocol is better to my purpose?
If the RNeasy kit is a good way to isolation RNA, how can I combine this protocol with Trizol protocol to obtain RNA from my samples that are incubated with Trizol at -80ºC? Or can I incubate the samples with RLT buffer at -80ºC or another compound?
Add 0.5ml (500µl) of 100% isopropanol to the aqueous phase, per 1ml TRIzol reagent used for homogenization. then Incubate for 10 minutes at room temperature. Centrifuge at 12000g X4°C X 10 minutes. Proceed to RNA wash.
Remove the supernatant from the tube, leaving only the RNA pellets.
Wash the pellet with 1ml of 75% ethanol per 1ml of TRIzol reagent used in the initial homogenization.
Note: the RNA can be stored in 75% ethanol for at least 1 year at -20°C, or at least for 1 week at 4°C.
Vortex the samples, then Centrifuge at 7500g X4°C X 5 minutes. Discard the wash.
Vacuum or air dry the RNA pellet for 5-10 minutes. Do not dry the pellet by vacuum centrifuge.
Note: do not allow the RNA to dry completely because the pellet can lose solubility
Proceed to RNA re-suspension.
Dear Sir Murtaza Ali , I have gone through the PDF file you attached with the reply to the question asked. What should I do, if I am dealing with a voucher specimen. For example: I collected live insects (very small size: about 400 micro meter) and store directly in 500ul TriZol. Should I crush samples in the same TriZol or take out and crush in liq.nit.
The protocol of TriZol requires measuring the sample size to use initial quantity of TriZol. I am unable to measure the number of cells in a full grown adult insect (very small size).
Dear Amit Kumar Shrivastava and Amanda Rocha can also answer from their experiences.
Thanks.
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