4 replies to this topic

[moljul]

I want to establish the isolation of circulating tumor cells from blood of patients!

how many cells do I have to harvest at least, to extract enough RNA for PCR analysis??
are there some special kits, to extract RNA from that small sample size?

thanks for recommendations

[epibio]

It would depend on the abundance of transcripts that you're trying to detect, but if you're working with a very small number of cells (<500) you'd need to amplify the RNA. An example of a kit for doing the extraction and amplification is Epicentre's MessageBOOSTER kit.

[moljul]

epibio, on 31 August 2010 - 11:18 AM, said:

It would depend on the abundance of transcripts that you're trying to detect, but if you're working with a very small number of cells (<500) you'd need to amplify the RNA. An example of a kit for doing the extraction and amplification is Epicentre's MessageBOOSTER kit.

thanks for recommendation. sounds interesting!
but, how can I quantify RNA to be sure to perform my following real time PCR reactions with the same amount of target RNA?

[epibio]

You wouldn't need to quantitate the absolute amounts, just normalize your qPCR using a housekeeping gene. However, if you really want to know the amount of cDNA, you could use a Nanodrop.

[DNAgeek]

200 or so cels would do.

A couple of kits to consider:
Agilent Absolutely RNA Nanoprep Kit or the PicoPure kit by Arcturus to isolate RNA from very small samples

Then you may like to converti it to cDNA, which is more stable than working with RNA. You can use kits like the ABI High-Capacity cDNA Reverse Transcription Kit.

If you need to pre-amplify your cDNA, you could use the Super SMART PCR cDNA Synthesis Kit by Clontech or the above kits may mention other kits.

Attached Files

•  Agilent Absolutely RNA kit.pdf   163.19K   147 downloads
•  PicoPure RNA isol kit manual.pdf   255.87K   156 downloads