I am going to perform whole genome expression profiling of different tumor cells of one breast tumor. I isolate these tumor cells by laser microdissection and extract RNA. The total RNA concentration does not exceed 10 ng (RIN 7-8). Are there expression microarrays that could work with low input of RNA? Could you please share with me the information and your experience?
Thank you very much! I think that whole transcriptome sequencing is good alternative to expression microarrays. However, I would like to firstly get information about microarray technology, because I already had experience with it. If expression array would be impossible using low input of RNA, I will try to apply RNA sequencing for my aims.
In our hands reliable profiles from as little as 0.5 ng total RNA (RIN surely below 6, we could not even measure!) can be obtained after preamplification with the RiboSPIA kit from Nugen (and Agilent arrays). We use "Ovation PicoSL WTA v2" but there is also a special kit available for FFPE material.
Dear Jochen, thank you! In my case, RNA is from frozen samples. What procedures do you perform using amplified RNA samples? Did you have experience with expression microarrays? What product do you finally get using Ovation PicoSL WTA v2? RNA or cDNA?
Ovation PicoSL WTA v2. We use total RNA from microdissected samples. There is an Application note from Nugen for Agilent Arrays. We follow this protocol/appnote.
Thanks a lot! I found the useful information on the Nugen website.
Does anybody have experience with the QuantiTect Whole Transcriptome Kit (Qiagen). What expression microarrays (Agilent, Illumina, Affymetrix etc.) are suitable for cDNA generated with this kit?
Hi Evgeny,
we developed a reliable protocol to extract RNA from fresh frozen laser microdissected mucosa samples for gene expression profiling. In our hands the Eberwine protocol worked sufficient for oligonucleotide array analysis. An in vitro transcription for 14 hrs from Ambions MessageAmp II-Biotin Enhanced kit yielded in an approximately 100-fold amplification of start RNA. Quality checks had been satisfactory.
PMID: 21830273, 21761304, 19039087
I hope this makes sense to you and helps.
Dear Benjamin,
Thank you very much for this information! Sure, it can resolve my problem.
Dear Mikael, thank you! Microfluidic PCR is a good approach to estimate expression in single cells, however, this method is restricted by number of the analysed genes. I'd like to perform full expression profiling of my samples.
By the way, does anybody have an experience with the nCounter® Analysis System for expression analysis? Is it possible to use low input of RNA?
Dear Rohan, I use LCM to isolate concrete tumor cells from frozen tumor. RIN from LCM samples varies from 6 to 8 (good), however, the RNA concentration is very low to perform expression profiling by microarray technology.
- Badges
- Science topic
- Similar topics
- Biological Science
- Genetics
- Gene Expression