3 replies to this topic

[Sayantani]

HI,
Can somebody suggest me a protocol for isolating mRNA as well as miRNA from CSF(cerebrospinal fluid)?I am absolutely naive in this field and not even getting much references about this things on web.I am just starting to work in this project .So somebody please help.And can you also clarify the problem of getting a band in 270 at nanodrop.And except nanodrop what are the other procedures of quantifyng isolated mRNA from sample?


Sayantani

[beth]

I would recommend TRIzol- you will get both large and microRNAs using this. You will lose small RNAs using spin cup protocols. You can buy specialised miRNA isolation kits, but I am pretty sure these are the same as TRIzol, but twice the price!

Not sure what you mean about the 270 band. I have used nanodrop after doing TRIzol and it seems fine. You could also try Ribogreen- takes a bit longer but works well. You just need a fluorescent plate reader, or you can read on a qPCR machine.

[UBClabbie]

Sayantani, on 12 December 2010 - 12:01 PM, said:

HI,
Can somebody suggest me a protocol for isolating mRNA as well as miRNA from CSF(cerebrospinal fluid)?I am absolutely naive in this field and not even getting much references about this things on web.I am just starting to work in this project .So somebody please help.And can you also clarify the problem of getting a band in 270 at nanodrop.And except nanodrop what are the other procedures of quantifyng isolated mRNA from sample?


Sayantani

I perform miRNA profiling using rt-qpcr and have found TRIzol is very reliable for isolating RNA and detecting miRNA. I find Trizol is gentle enough to isolate pure RNA. just make sure u don't remove the whole aqueous phase, leave about 150ul of it to reduce contamination.
You could use other methods to isolate small RNA... but what exactly are u using the miRNA for?
my advice is to isolate using TRIzol and then possible use an additional kit to further isolate small RNA.
hope this helps

ps. i use a regular spectrophotometer to measure RNA concentration in my samples. i usually read using a diluted sample in a cuvette nanodrop is great for conserving sample but only if neccessary. i find its finicky sometimes...

Edited by biotechgirl, 03 January 2011 - 04:03 PM.

[UBClabbie]

Sayantani, on 12 December 2010 - 12:01 PM, said:

HI,
Can somebody suggest me a protocol for isolating mRNA as well as miRNA from CSF(cerebrospinal fluid)?I am absolutely naive in this field and not even getting much references about this things on web.I am just starting to work in this project .So somebody please help.And can you also clarify the problem of getting a band in 270 at nanodrop.And except nanodrop what are the other procedures of quantifyng isolated mRNA from sample?


Sayantani

check out this post. Limey, who works w/ nanodrop, says readings at 270 may indicate a contamination problem http://www.protocol-...ion-from-serum/