I would like to isolate total RNA from single cells for qPCR (or even sequencing later on). What protocol or kit should I use to obtain good quality RNA with minimum loss?? I've seen there is a kit "PicoPure® RNA Isolation Kit" but I'd like to know if there are other suggestions before buying such and expensive kit..
Actually, after reading the paper and couple more about direct cell lysis and qRT-PCR, I saw that they all did one step qRT-PCR, without synthesising global cDNA first. I actually would like to synthesise cDNA first and use it to detect RNA levels of several genes, not just one.
I saw that there are kits for cDNA synthesis from single cells (and also directly from cell lysates) and they are vastly used in most papers I've read, but, again, they are quite expensive. I compared the components and recipes of the buffers of SuperScrip III RT and SuperScript III CellsDirect cDNA Synthesis System (Invitrogen) and they look exactly the same.. Do you think it would be necessary to use a kit instead of going ahead with the usual procedures for cDNA snthesis and qPCR?
Dear Dr. Mona,
I hope this is going to help you.
RNA isolation kit utilizes a rapid single-step extraction (guanidine thiocyanate/phenol/chloroform extraction) to isolate undegraded total RNA; the kit contains enough reagents to isolate total RNA from at least 7g of tissue.
Content: Denaturating solution, phenol saturated with H2O, β-mercaptoethanol, chloroform:isoamyl alcohol, 2 M sodium acetate, isopropanol, and instruction manua.
RNA isolation kit
BioUltra, for molecular biology
Synonym: Ribonucleic acid isolation kit
Best Regards,
Ahmed Hussein,PhD
Professor of Microbiology and Molecular Biology,
Institute of Graduate Studies and Research,
Department of Biotechnology
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