RNA Analysis with Low Copy Number Templates

Together, Ambion and Applied Biosystems (AB) provide researchers with a complete family of products that takes experiments from sample preparation through target detection and analysis.

For limited samples and small sample sizes, RNA can be extracted from numerous small tissue samples with one of several nucleic acid isolation kits from Ambion (see table). Quantitative analysis of gene expression can then be assessed using RT-PCR reagents and instruments from Applied Biosystems (Figure 1).

Figure 1. Family of Ambion and Applied Biosystems Products For Analyzing RNA with Low Copy Number Templates.
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RNA Isolation

Ambion, The RNA Company®, has a complete line of RNA isolation products that addresses a wide range of samples and applications.

MELT™ Total Nucleic Acid Isolation System (patent pending) provides hands-free tissue digestion that takes only minutes—no grinding or use of a Polytron® homogenizer required—making MELT ideal for processing samples for downstream high throughput amplification.

MagMAX™-96 Total RNA Isolation System is optimized for high throughput isolation of RNA from 25 cells to 2 x 106 cells, as well as small plant and mammalian tissue samples (up to 10 mg). This magnetic bead-based technology binds RNA more efficiently than glass fiber filter methods, resulting in higher RNA yields.

RNAqueous®-Micro Kit is optimized for the purification of total RNA from micro-sized samples such as 10 to 5 x 105 cultured cells, as few as 10 microdissected cells, or up to 10 mg tissue. Samples are solubilized in a powerful chaotropic solution, and RNA is purified on a glass fiber filter cartridge specifically designed for very small samples. A simple modification of the protocol allows recovery of both large and small RNA species including tRNAs, 5S rRNA, and microRNAs.

RNAqueous®-4PCR Kit is designed to isolate RNA free of genomic DNA contamination from very small samples such as needle biopsies and small numbers of isolated cells (as little as 1 mg of tissue or 100 cells).

Reverse Transcription

The High-Capacity cDNA Reverse Transcription Kits for 200 and 1000 reactions contain reagents for reverse transcription (RT) of total RNA to single-stranded cDNA. While designed for short or long term archival of cDNA, the kit yields very quantitative amplification of total RNA from 0.02 to 2 mg of total RNA.The resultant cDNA is ideal for:

  • Short-term and long-term archival storage
  • Quantitative PCR
  • Conversion to cRNA

NEW--Sample Preamplification

Before quantitative, real-time PCR, use the TaqMan® PreAmp Master Mix Kit to enrich cDNA for specific gene targets. This preamplification process is uniform and does not introduce bias, so this kit is ideal for rare or small samples (for example, laser capture microdissection, formalin-fixed paraffin-embedded tissues, needle biopsies, and single cells).

Target Amplification

Pre-designed, pre-optimized TaqMan® Gene Expression Assays are a comprehensive collection of over 700,000 probe and primer sets for quantitative gene expression analysis of human, mouse, rat, Arabidopsis, and Drosophila genes. These assays let you quickly, easily, and affordably perform precise gene expression studies using real-time PCR.

Detection

The Applied Biosystems 7300, 7500, 7500 Fast, and 7900HT Real-Time PCR systems offer you a portfolio of powerful and versatile platforms for real-time PCR detection. The 7500 Fast system’s high speed performance delivers results in 30 minutes.


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