Hello!

I was wondering is it possible to freeze monocytes and lymphocytes in order to amplify mRNA later, when I collect the samples from all the patients included in the study

est regards

Yes that should work. I've heard of people using products such as RNAlater as well to help preserve the RNA.

http://www.ambion.com/techlib/resources/rnalater/

Thank you very much for Your relevant post.

Best Regards

It's common to use RNALater for tissue samples, but if you have individual cells it might be better to do the RNA prep whilst the cells are fresh and maybe spend the extra 90 min to do the cDNA synthesis. This would then be stable and simple to go back to for PCR if that's what you plan to do.

Hello,
I think the best way to store yr lymphocyte is to suspend yr cell pellet in Trizol which has GITC and Phenol that keeps the RNA intact even if you extract RNA after year or more than that.

Firoz

boolean:

Here are some references that may help...

Good Luck!



S C C Wong, E S F Lo, and M T Cheung. An optimised protocol for the extraction of non-viral mRNA from human plasma frozen for three years. J Clin Pathol. 2004 July; 57(7): 766768.
doi: 10.1136/jcp.2003.007880.

Abstract:

Aims: To detect non-viral mRNA in human plasma that has been frozen for three years using a new protocol.

Methods: Plasma from 15 patients with colorectal cancer and 10 normal subjects was separated and frozen with Trizol at −80°C for three years. As a control measure, plasma from 10 of the 15 patients was separated using the same protocol but no Trizol during storage. After three years, all samples were extracted using Trizol and RNeasy before the reverse transcriptase polymerase chain reaction was performed to detect non-viral β catenin mRNA. In addition, extraction of three plasma samples by Trizol or RNeasy independently was carried out for comparison.

Results: β Catenin mRNA was detected in all 15 patient plasma samples and only one of the 10 normal subjects. In contrast, no β catenin mRNA was found in the control and patient samples that were independently extracted by Trizol and RNeasy kit.

Conclusions: This new protocol is a reliable method for extracting non-viral mRNA from the plasma of patients with cancer after longterm storage for three years. Extractions using Trizol and RNeasy kits independently could not isolate mRNA with sufficient quantity and quality for detection.


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Ambion Online Reference - The Basics: RNA Isolation

Overview:
Obtaining high quality, intact RNA is the first and often the most critical step in performing many fundamental molecular biology experiments, including Northern analysis, nuclease protection assays, RT-PCR, RNA mapping, in vitro translation and cDNA library construction. To be successful, however, the RNA isolation procedure should include some important steps both before and after the actual RNA purification. The following article discusses various RNA isolation procedures and ways of increasing RNA yields.


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University of Toronto Mississauga (Mississauga, ON) - Trizol Method



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University of Toronto Mississauga (Mississauga, ON) - Troubleshooting: RNA Isolation with TRIZOL Reagent Problems


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Amanda B. Hummon, Sharlene R. Lim, Michael J. Difilippantonio, and Thomas Ried. Isolation and solubilization of proteins after TRIzol extraction of RNA and DNA from patient material following prolonged storage . BioTechniques April 2007 Volume 42, Number 4: pp 467-472.

Abstract:
A systems approach is being applied in many areas of the biological sciences, particularly in cancer research. The coordinated, simultaneous extraction of DNA, RNA, and proteins from a single sample is crucial for accurate correlations between genomic aberrations and their consequences on the transcriptome and proteome. We present an approach to extract and completely solubilize up to 98% of the total protein recovered from archived samples following TRIzol isolation of RNA and DNA. We also demonstrate using polyacrylamide gel electrophoresis (PAGE) and Western blot analysis that the proteins, representing both a wide molecular weight range and some posttranslational modifications, such as protein phosphorylation, remain stable in phenol-ethanol for up to 3 years at -20°C.


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Your additional info is extemelly useful.

Thank you