Submitted to: Journal of Cotton Science
Publication Type: Peer Reviewed Journal
Publication Acceptance Date: September 15, 1997
Publication Date: N/A
Interpretive Summary: Isolation of high quality of protein, RNA and DNA are difficult in cotton plants due to presence of high levels of phenolic compounds. Some methods are available currently for proteins, RNA and DNA extraction in cotton using fresh tissues. However, it may be difficult to analyze a large number of samples using fresh tissues. A practical solution to this problem is to develop suitable procedures for storing tissues and improving the methods of extraction of protein and nucleic acids. We describe here methods comparing freshly collected cotton samples stored at -50C and freeze-dried samples for extraction of high yield and quality of protein, RNA and DNA. Our results showed that both fresh frozen and freeze-dried samples provided good qualities and high yield of DNA. The DNA was readily digestible with restriction enzyme and thus suitable for RFLP analysis. The DNA was amplified with gene-specific primer, accordingly appropriate for PCR analysis. However, our results indicated that only fresh frozen samples at -50C provided good quality of RNA. Our results showed that RNA from fresh frozen samples in agarose gel had two distinct bands of 28S and 18S ribosomal RNA. In vitro translation using RNA from the fresh frozen root samples showed the presence of variable sizes of protein bands. This indicated that extracted RNA had good quality of mRNA, which can be used for gene cloning and other purposes. Both fresh frozen samples at -50C and freeze-dried samples provided good quality of proteins of variable sizes as separated in SDS-PAGE analysis.
Technical Abstract: We tested whether lyophilized (i.e. freeze-dried) cotton tissue was suitable as a source for DNA, RNA, and protein isolation. Extraction methods, which we have found effective for nonfreeze-dried (NFD) tissues, are presented here and in addition were tested on freeze-dried (FD) leaf and root tissue of cotton. Our results showed that FD leaf tissue from either greenhouse cultured or field grown cotton yielded high moelcular weight genomic DNA. The DNA was suitable for restruction enzyme digestion and as template for PCR amplification. In contrast, freeze-drying led to complete degradation of RNA in leaf and root tissue. Total proteins of leaves and roots were unaffected by freeze-drying based on comparison of polypeptide profiles by denaturing polyacrylamide electrophoresis. While these results rule out FD tissue as a source for RNA isolations, the ability to freeze-dry, powder, and efficiently store voluminous tissue samples for later use in DNA and protein isolation could be of great benefit to laboratories involved in cotton molecular genetics.