Hello Sadegh,

There are actually several options. We have worked many times from small numbers of cells (down to single cells). One option is to go direct from the cell. For example, we published a technique way back in 2002 that works quite well (http://www.nature.com/nbt/journal/v20/n9/full/nbt729.html). The only drawback really is that it is very 3'-biased and so some microarrays don't work great with it.

Alternatively there are several commercial kits now that work well. You can check out Nugen's WT-Ovation One-Direct kit, or Clontech's SMARTer Ultra Low input RNA kit which was designed to work with Illumina Sequencing.

Neil

@ Dear Neil;

I have about 50 cells from diseased cases and also normals. Because every cell can excist in different cell cycle stage, I want to pool them and then extract mRNA. Cells are microdissected. I wonder if you can help me how validate microarray results. How I can make sure that results are showing what happens in a cell's transcriptome? How can I make sure about unbiased amplification before microarray?

thanks

There are many ways where you can complete your goal - 1) through PCR or 2) through plasmid (vector) replication or 3) bacteriophage.

PCR reaction will allow to amplify the naked cDNA in number of time as you wanted while in plasmid-vector replication you have to transfer plasmid into bacteria and multiplication of plasmid into bacteria (just like you have prepared cDNA library).

@Ankush Jadhav

I want to amplify whole cDNA library for microarray.

Hi Sadegh

Try to adapt a method that we succesfully used:

Biotechniques. 1993 Oct;15(4):654-6, 658-9.

Construction of subtractive cDNA libraries from limited amounts of mRNA and multiple cycles of subtraction.

López-Fernández LA, del Mazo J.

Hi Sadegh,

I have the similar situation and here is our publication and may be useful for you to resolve the problem. Our amplification method works well with Agilent microarray. Stephen

Data Development of a porcine embryo specific microarray

Also, we used the SMART technology, and we amplified single cell, or a very small amount of total RNA, only give more amplification cycles to double-stranded cDNA.

Juan Jimenez Bremont