TRI Reagent^®RT

 

TRI Reagent^® RT is an upgraded version of the single-step method of RNA isolation. TRI Reagent RT is based on the phase

separation method using phenol and guanidine thiocyanate. This patent-pending reagent substantially improves the quality

of isolated RNA. Unlike the previous single-step methods, TRI Reagent RT isolates RNA that is free of DNA contamination.

The purity of RNA was tested by a 35-cycle PCR. Thus, no DNase treatment is necessary to use the isolated RNA in RT-PCR.

Also, the isolated RNA solubilizes more quickly and easily as compared with the older single-step methods.

TRI Reagent^® RT is used for RNA isolation from tissues, pelleted cells and cells grown in monolayer.

Fifty milliliters is sufficient to process 50 samples, each containing 50 mg of tissue.

Expected yields range from 50 – 700 µg of RNA per sample, depending upon the tissue source.

 

TRI Reagent^® RT-Blood is designed for use with whole blood and plasma.

Fifty milliliters is sufficient to process 65 samples of 0.25 ml each.

 

TRI Reagent^{® RT-Liquid Samples} is used for cell suspensions and other liquid samples.

Fifty milliliters is sufficient to process 65 samples of 0.25 ml each.

 

 

Total RNA was isolated from rat kidney (50 mg tissue / ml TRI Reagent RT) and examined for RT-PCR acceptability and DNA contamination.

RNA or cDNA was not DNAse treated prior to reactions. (A) RNA was reverse transcribed using 1.0, 0.3, or 0.1 mg RNA template.

PCR was performed using 2 ml of cDNA product as template, for 35 cycles in a 20 ml reaction volume; 16 ml of reaction

product was loaded per lane. Genomic DNA was used for positive controls; water template was used in negative controls.

Both high copy (GAPDH, PGK) and low copy (cfos) genes were successfully amplified. (B) RT was performed using 1.0 mg

RNA template. Positive and negative RT product was amplified for 35 PCR cycles. Negative RT verified no DNA contamination

of RNA samples. Amplification products were electrophoretically separated in a 2% Super Agarose gel with ethidium bromide staining.

Total RNA was isolated from rat kidney (50 mg tissue / ml TRI Reagent RT) and examined for RT-PCR acceptability and DNA contamination.

RNA or cDNA was not DNAse treated prior to reactions. Four samples each were amplified for 40 cycles following positive or negative RT

amplification (1.0mg RNA template; 2 ml reaction volume as PCR template). No amplification from negative RT samples verified

RNA was free of DNA contamination without DNAse treatment. A high copy gene, GAPDH, was used for this verification.