TY - JOUR
T1 - Efficient extraction and quantitative determination of nanogram amounts of cellular RNA
AU - Schultz, Richard M.
AU - Wassarman, Paul M.
N1 - Funding Information:
This research was supported by grants from the NIH, the NSF, and the Rockefeller Foundation. The authors thank Dr. Charles C. Richardson for helpful discussions and Dr. Neville Kallenbach for donating [y-3’P]ATP on numerous occassions.
PY - 1980/5/15
Y1 - 1980/5/15
N2 - An assay for quantitating nanogram amounts of cellular RNA is described. RNA is efficiently extracted from cells, using RNA-free DNA as carrier, by conventional chloroform: phenol procedures and the nucleic acids are precipitated with ethanol. Isolated RNA is hydrolyzed by RNase T2 to ribonucleoside 3′-monophosphates which in turn are converted to 5′-32P-labeled ribonucleoside 3′,5′-diphosphates in the presence of T4 polynucleotide kinase and [γ-32P]ATP. Radiolabeled products are separated from remaining [γ-32P]ATP by chromatography on polyethyleneimine-cellulose, located by autoradiography, excised from the chromatogram, and subjected to liquid scintillation counting to quantitate the amount of RNA. Using mouse liver ribosomal RNA as a standard, the assay is linear over a range of 0 to 64 ng of RNA. The assay has been used to determine the amount of RNA in fully grown mouse oocytes arrested at the dietyate stage of first meiotic prophase. Each oocyte contains 0.61 ± 0.05 ng of RNA and only 25 oocytes have been used for such assays.
AB - An assay for quantitating nanogram amounts of cellular RNA is described. RNA is efficiently extracted from cells, using RNA-free DNA as carrier, by conventional chloroform: phenol procedures and the nucleic acids are precipitated with ethanol. Isolated RNA is hydrolyzed by RNase T2 to ribonucleoside 3′-monophosphates which in turn are converted to 5′-32P-labeled ribonucleoside 3′,5′-diphosphates in the presence of T4 polynucleotide kinase and [γ-32P]ATP. Radiolabeled products are separated from remaining [γ-32P]ATP by chromatography on polyethyleneimine-cellulose, located by autoradiography, excised from the chromatogram, and subjected to liquid scintillation counting to quantitate the amount of RNA. Using mouse liver ribosomal RNA as a standard, the assay is linear over a range of 0 to 64 ng of RNA. The assay has been used to determine the amount of RNA in fully grown mouse oocytes arrested at the dietyate stage of first meiotic prophase. Each oocyte contains 0.61 ± 0.05 ng of RNA and only 25 oocytes have been used for such assays.
UR - http://www.scopus.com/inward/record.url?scp=0019325416&partnerID=8YFLogxK
U2 - 10.1016/0003-2697(80)90083-4
DO - 10.1016/0003-2697(80)90083-4
M3 - Article
C2 - 6160786
AN - SCOPUS:0019325416
SN - 0003-2697
VL - 104
SP - 328
EP - 334
JO - Analytical Biochemistry
JF - Analytical Biochemistry
IS - 2
ER -