, . Methods Useful in Genetics and Oncology
Sample Preparation
Purification
of Poly-(A) RNA
and eDNA Synthesis
mRNA Capture Kit (Cat. 1787896; Roche Molecular Biochemicals, USA)
1st Strand cDNA Synthesis Kit for RT-PCR (Cat. 1483188; Roche Molecular Biochemicals)
LightCycler DNA Master SYBR Green I Kit (Cat. 2015099; Roche Molecular Biochemicals)
Betaine 5 M (Cat. B0300; Sigma, USA)
TNF-a cDNA clone (American Type Culture Collection Cat. 39918)
IL-l~ cDNA clone (American Type Culture Collection Cat. 581769)
IL-6 cDNA clone (American Type Culture Collection Cat. 567519)
~-actin cDNA (Cat. 9800-1, Clontech Laboratories, USA)
Procedure
Human myocardial biopsy tissue samples (-2-3 mg and >50 mg) from failing
hearts were collected postmortem after heart transplantation, flash frozen in liquid nitrogen, and stored at -80°C. The frozen samples were thawed in 1 ml of Trizol or RNA-STAT solution and immediately homogenized using a Polytron 3000
homogenizer at 30,000 rpm for 30 s. After homogenization, the total RNA was
extracted and precipitated according to the manufacturer's protocol. Isolated
RNA was immediately stored at -80°C until use. Due to the minute amount of
total RNA from myocardial biopsy samples (-2-3 mg), the concentration of RNA
was not spectrophotometrically determined. However, when the tissue samples
were of a larger size (>50 mg), the concentration and purity of RNA preparations
were measured at A26o, A2so and A320 using a Beckman 540 spectrophotometer.
Poly-A RNA from the total RNA was captured using biotin-labeled oligo-(dT}zo
(mRNA Capture Kit) according to the manufactures protocol. Following capture,
the poly-A RNA was immobilized by transferring the captured RNA to straptavidin-coated microfuge tubes. After immobilization, the solution was removed
from the straptavidin-coated tubes and the tubes were washed three times and
stored at -80°C. First-strand cDNA synthesis (lst Strand cDNA Synthesis Kit for
RT-PCR) was directly performed on the immobilized poly-A RNA according to
the manufacturer's protocol. Briefly, oligo-p(dN)6 primer was added to the straptavidin-coated tubes containing immobilized poly-A RNA and reverse transcribed into single-stranded cDNA. Because of the use random hexamer for first
strand cDNA synthesis, multiple copies of the same mRNA will be made as short
single-stranded cDNA segments from different locations, and these nascent
cDNA segments can be freed from the template by denaturation. Thus, the reaction was terminated by denaturing the cDNA products at 95°C, and the synthesized cDNA products were removed from the straptavidin-coated tubes and
stored at -20°C for quantitative PCR. Later, the straptavidin-coated tubes containing the immobilized poly-A RNA were washed again to remove the residual
cDNA and stored at -80°C for future use. We have used the immobilized poly-A
RNA three times for repeated synthesis of cDNA, and quantitative PCR yielded
identical results each time. Additionally, we have also generated single-stranded
Sample Preparation
Purification
of Poly-(A) RNA
and eDNA Synthesis
mRNA Capture Kit (Cat. 1787896; Roche Molecular Biochemicals, USA)
1st Strand cDNA Synthesis Kit for RT-PCR (Cat. 1483188; Roche Molecular Biochemicals)
LightCycler DNA Master SYBR Green I Kit (Cat. 2015099; Roche Molecular Biochemicals)
Betaine 5 M (Cat. B0300; Sigma, USA)
TNF-a cDNA clone (American Type Culture Collection Cat. 39918)
IL-l~ cDNA clone (American Type Culture Collection Cat. 581769)
IL-6 cDNA clone (American Type Culture Collection Cat. 567519)
~-actin cDNA (Cat. 9800-1, Clontech Laboratories, USA)
Procedure
Human myocardial biopsy tissue samples (-2-3 mg and >50 mg) from failing
hearts were collected postmortem after heart transplantation, flash frozen in liquid nitrogen, and stored at -80°C. The frozen samples were thawed in 1 ml of Trizol or RNA-STAT solution and immediately homogenized using a Polytron 3000
homogenizer at 30,000 rpm for 30 s. After homogenization, the total RNA was
extracted and precipitated according to the manufacturer's protocol. Isolated
RNA was immediately stored at -80°C until use. Due to the minute amount of
total RNA from myocardial biopsy samples (-2-3 mg), the concentration of RNA
was not spectrophotometrically determined. However, when the tissue samples
were of a larger size (>50 mg), the concentration and purity of RNA preparations
were measured at A26o, A2so and A320 using a Beckman 540 spectrophotometer.
Poly-A RNA from the total RNA was captured using biotin-labeled oligo-(dT}zo
(mRNA Capture Kit) according to the manufactures protocol. Following capture,
the poly-A RNA was immobilized by transferring the captured RNA to straptavidin-coated microfuge tubes. After immobilization, the solution was removed
from the straptavidin-coated tubes and the tubes were washed three times and
stored at -80°C. First-strand cDNA synthesis (lst Strand cDNA Synthesis Kit for
RT-PCR) was directly performed on the immobilized poly-A RNA according to
the manufacturer's protocol. Briefly, oligo-p(dN)6 primer was added to the straptavidin-coated tubes containing immobilized poly-A RNA and reverse transcribed into single-stranded cDNA. Because of the use random hexamer for first
strand cDNA synthesis, multiple copies of the same mRNA will be made as short
single-stranded cDNA segments from different locations, and these nascent
cDNA segments can be freed from the template by denaturation. Thus, the reaction was terminated by denaturing the cDNA products at 95°C, and the synthesized cDNA products were removed from the straptavidin-coated tubes and
stored at -20°C for quantitative PCR. Later, the straptavidin-coated tubes containing the immobilized poly-A RNA were washed again to remove the residual
cDNA and stored at -80°C for future use. We have used the immobilized poly-A
RNA three times for repeated synthesis of cDNA, and quantitative PCR yielded
identical results each time. Additionally, we have also generated single-stranded