Study on differentially expressed mRNAs in lactating and nonlactating buffaloes
Singh, Satinder (2004) Study on differentially expressed mRNAs in lactating and nonlactating buffaloes. Masters thesis, College of Veterinary Science and Animal Husbandry, Anand Agricultural University, Anand, Gujarat, INDIA.
Full text available as:
Gene expression in mammary cells lies at the heart of the mechanisms controlling milk synthesis and, therefore, milk composition. The recent development of differential display technology solves many biological questions not previously amenable to research. Present study was planned with the objective of standardizing the techniques for, total RNA extraction from udder tissue, differential display, eluting differentially expressed cDNAs and to study the differentially expressed mRNAs in lactating and non lactating buffaloes. A total of six udder tissue samples were taken for the study i.e. two each from: buffalo heifers, lactating buffaloes and dry buffaloes. Two methods were compared for total RNA extraction from tissue samples: TRIZOL based protocol for total RNA extraction from mammalian tissue and RNeasy protect mini kit. Total RNA extracted with TRIZOL had comparatively high yield (20-25%) and purity among the two RNA extraction methods. With QIAGEN’s RNeasy protect mini kit, per sample RNA extraction proved approximately 4 times (Rs.406/-) costlier than TRIZOL reagent based method (Rs.105/-). However, processing the sample with QIAGEN’s RNeasy protect mini kit (15 min) was faster then with TRIZOL reagent method (55 min). First strand cDNAs were synthesized by using two different reverse transcriptase preparations (1) First-strand cDNA synthesis using Omniscript reverse transcriptase kit (QIAGEN) for two tube RT-PCR and First-strand cDNA synthesis using SUPERSCRIPT II (Gibco BRL). PCR was carried out with three DDRT primers and three random primers. Positive amplicons were tested on polyacrylamide gel. Twenty five differentially regulated cDNA fragments were then recovered from polyacrylamide gels by “crush and soak" method. Out of that 17 fragments were reamplified for further analysis. The integrity of bands was reconfirmed and amplicons then eluted from agarose using Qiagen’s Gel Extraction kit and with organic solvent extraction method. The QIAGEN’s gel extraction kit eluates showed significantly higher yield (18–20%) as compared to the organic solvent extracted ones. Also the kit method takes quite less time (25 min) for complete process of elution as compared to the organic solvent extraction method (1 hr). But the organic solvent extraction method proved much cheaper (70–80 times) to that of QIAGEN’s gel extraction, where each sample processing expenditure came out to be Rs. 100/-. The eluted products were again subjected to PCR and purity of bands analyzed. The bands were subsequently subjected to sequencing. One product (3L31C4) was apparently generated by amplification with single 10-mer priming at 5’ and 3’ positions. Two showed (1H31C7 and 3L31C4) sequence similarity with Oryza sativa genomic DNA. cDNA 1H23A4 showed full sequence similarity with Mus musculus chromosome 11. DD fragments, 3D31C3, 3L38D14 and 2L31C6 did not show full sequence similarity match with any of the identified gene sequences in public databases.
Archive Staff Only: edit this record