Detailed Reference Manual (English Version Only)


Application Example

Figure: Analysis of the total RNA fraction isolated with the DNA + RNA + Protein kit. Total RNA preparation from HEP G2 (liver cancer) cell cultures. An optional DNase digestion was not performed. Analysis was performed with an Agilent Bioanalyzer ®.

• Between 100 and 200 ng of high quality total RNA were obtained per column (recommended value: max. 100 ng RNA per column, pictured: 177 ng RNA per column).
• Analysis of triplicate extractions gave RNA integrity numbers (RIN) between 9.50 and 10.00 (pictured: 9.90) for all analyzed samples.
• The rRNA ratio (28S to 18S) was always >2, as expected for high quality RNA preparations (pictured: ~2.5).
• No visual traces of genomic DNA are detectable (if visible, genomic DNA contamination would be detectable as ~ 20 kb sized band). Purified total RNA was free from contaminating DNA, even without any DNase digestion steps.

Absence of contaminating genomic DNA at detectable levels was experimentally confirmed by PCR using the human beta-actin gene specific primers ß-Ac-fwd(5‘-CGG ATG TCC ACG TCA CAC TT-3‘), ß-Ac-rev (5‘-GTT GCT ATC CAG GCT GTG CT-3‘) and the PCR amplification parameters 95°C/120 s / 30x [94°C/30s – 60°C/30s – 72°C/60 s] / 72°C/600 s, as described elsewhere (Adjaye et al. (1999) Gene 237: 373–383). Note: This PCR assay allows as well to differentiate between amplification from genomic DNA templates (~ 565 bp amplicon) and cDNA templates (~ 470 bp amplicon), thus enabeling post-RT detection of genomic DNA contaminations in cDNA samples.
Results: (1) No product obtained using purified RNA as template; (2) specific product obtained with genomic template DNA as positive control. Positive control DNA template: DNA fraction from the same sample obtained following the DNA purification steps during this extraction. The sensitive PCR assay confirmed the absence of contaminating DNA traces from purified RNA.
Purified total RNA was further used for cDNA synthesis and subsequent qPCR / RealTime PCR analyses with excellent results.

Protocols available for RNA extraction from

• human and animal tissue samples;
• plant tissue samples;
• bacterial cells;
• yeast cells;
• cell culture (human and animal cell lines).

Quality Characteristics

• Simultaneous, fractionated isolation of DNA, RNA and protein from one single sample.
• Protocols available for samples from human or animal tissue, from plant, bacteria and cell cultures.
• Completely phenol-chloroform free purification procedure - no damage or loss of nucleic acids due to attack of free radicals in organic compounds.
• Separates RNA completely from DNA - no DNase digestion required.
• High yield, even with small sample amounts - ideal for scarce sample material (e.g. cell culture monolayers, biopsies).

Related Articles

• dART cDNA Synthesis Kit with all required and specifically adjusted components for Reverse Transcription
• Reverse Transcriptases
• Ribonuclease Inhibitor
• Ribonuclease H ( E.coli ) for selective RNA digestion from DNA/RNA hybrids only
• SG qPCR Master Mixes (2x) for qPCR / RealTime PCR

Additional Resources

 RT Logsheet

 PCR Logsheet

 PCR Master Mix Logsheet

 qPCR Logsheet