Top Five Tips for Working With Limited Samples

As RNA analysis techniques, such as reverse transcription followed by real-time PCR and microarray analysis, become increasingly sensitive, researchers are looking to analyze ever smaller samples. There are numerous strategies for handling micro-sized samples. Some rely on techniques that have been available for years, but newer reagents and techniques have greatly expanded the tool set for research that relies on limited samples. No matter what the source—small tissue pieces, low cell numbers, microdissected cells—these tips will help obtain the maximum data from limited samples.

1. Use an Appropriate RNA Isolation Kit
Make certain you are getting maximum yield by using a kit developed for RNA recovery from limited samples:
• The Ambion RNAqueous®-Micro Kit efficiently recovers RNA from small amounts of a sample—as few as 10 to 500,000 cultured cells, 10 microdissected cells, or up to 10 mg tissue.
• The Ambion RecoverAll™ Total Nucleic Acid Isolation Kit for FFPE Tissues utilizes optimized protease digestion conditions to release maximal amount of nucleic acid from formaldehyde- or paraformaldehyde-fixed, paraffin-embedded tissues. (See article, Your Data: Identifying Cancer-Related mRNA Signatures in FFPE Patient Samples, for an example of use of this kit.)

2. Use a Suitable Coprecipitant
For quantitative recovery of low concentrations of RNA (ng/mL) in alcohol precipitations, an inert coprecipitant (e.g., glycogen, yeast RNA, or linear acrylamide) should be used. Linear acrylamide and DNase-treated glycogen are the coprecipitants of choice when the RNA will be used in reverse transcription-PCR (RT-PCR) because they do not contain contaminating DNA. Yeast RNA and untreated glycogen could introduce nucleic acid contamination into samples, potentially skewing RT-PCR results. After precipitation, avoid complete drying of the RNA pellet, because coprecipitants can make RNA difficult to resuspend.

3. Skip RNA Purification
One can now directly perform RT-PCR from cultured cells without first isolating RNA. The TaqMan® Cells-to-CT™ family of kits enables the lysis of cultured cells with removal of genomic DNA while preserving RNA integrity. The cell lysates can then be used directly in RT-PCRs and exhibit the same sensitivity and specificity as purified RNA in real-time PCR.

The TaqMan Gene Expression Cells-to-CT Kit is designed for use with any TaqMan Gene Expression Assay. It is ideal for both small samples and for any research involving analysis of gene expression in cultured mammalian cells.

The TaqMan MicroRNA Cells-to-CT Kit is the first kit to offer a complete workflow for miRNA analysis, and has been developed with state-of-the-art TaqMan MicroRNA RT reagents and assays.

4. Amplify RNA for Microarray Analysis
Most microarrays require 5–20 µg of RNA/slide for sample labeling and hybridization. Linear amplification of RNA using the Ambion MessageAmp™ Premier and MessageAmp III RNA Amplification Kits produces sufficient RNA for analysis and maintains representation of the starting mRNA population from as low as 20 ng input total RNA. These next-generation RNA amplification kits simultaneously amplify and add a biotin label to the resulting aRNA; they are designed to prepare samples for gene expression analysis on Affymetrix® GeneChip® arrays.

5. Minimize RNA Degradation
Traditional staining protocols involve exposing laser capture microdissection (LCM) samples to aqueous solutions which can cause RNA degradation, presumably due to reactivation of endogenous RNases. The Ambion LCM Staining Kit avoids exposing tissue sections to pure water, which results in superior staining of frozen sections while preserving RNA quality.

Tissues prepared for LCM using the LCM Staining Kit yield high quality RNA suitable for qRT-PCR and microarrays.The kit has been tested with a variety of tissue types from mouse (brain, liver, spleen, and kidney) and human (ovarian tumor, uterine tumor, lymphoma, small intestine, colon and breast).