ON TESTIS AND EPlDlDYMlS OF RATS - Pondicherry University ...

centrifuged at 3,500 g for I5 min on a Remi R8C Centrifuge. The supernatant was
discarded and the pellet was resuspended in 2.5 mL of 10% TCA. It was then
centrifuged at 3,500 g for 15 min. The supernatant was discarded and the pellet was
resuspended in 5 mL of 95% ethanol. Extraction of lipids was completed with
vortexing in 3:l ethanol ether mixture for 5 min and then centrifugation at 3,500 g for
I5 min.
The pellet was re-extracted with 5 mL of 95% ethanol. The supernatants were
discarded. The pellet was resuspended in 3 mL of 5% perchloric acid (PCA) and was
heated for I5 min in a hot water bath at 90°C. After cooling the tubes were
centrifuged at 3.500 g for 15 min. The extraction of nucleic acids was completed by
resuspending the pellet in 5% PCA and centrifugation at 3,500 g for 10 min. The
supcrnalant fluids wcre pooled and were used for DNA and RNA determinations.
The residual pellet was dissolved in 0.1 N sodium hydroxide after suitable dilution.
3.15.2 Determination of DNA
DNA was determined by diphenylamine colour reaction following the method
of Burton (1956). The tubes containing 1 mL nucleic acid extract, 2 mL of IN
perchloric acid and 2 mL of diphenylamine reagent (see Appendix 1 .lo) were kept in
a boiling water bath for 20 min. The samples were cooled and the colour developed
was read at 600 nm on a Systronics Spectrophotometer. A standard curve was
prepared by using known concentrations of calf thymus DNA.
3.15.3 Determination of RNA
RNA was determined by orcinol reaction method (Schneider, 1957). The
tubes containing 2 mL nucieic acid extract and 3 mL of orcinol reagent (see Appendix
I. I I) wcre kept in a boiling water bath for 20 nlin. The samples were cooled and the
colour developed was read at 665 nm on a Sysuonics Spectrophotometer. A standard
curvc was prcparcd by using known concentrations of yeast RNA.