LAB 4

Preparation of Yeast RNA and Synthesis of cDNA

Name: Mubarak Alhassan

Introduction

Obtaining high quality, intact RNA is the first and often the most critical step in performing many fundamental molecular biology experiments, including Northern analysis, nuclease protection assays, RT-PCR, RNA mapping, in vitro translation and cDNA library construction. To be successful, however, the RNA isolation procedure should include some important steps both before and after the actual RNA purification. In this Lab experiment, we learn ways to perform RNA isolation in ways that produce RT-PCR competent RNA.
MATERIALS AND METHODS

Growth of Yeast and RNA Isolation Before the class begins, the TA grow yeast cells in YEPD to mid exponential phase (OD600 = about .200 - .4) Total RNA was isolated using TES solution (phenol, and sodium dodecyl sulfate (SDS) to reduce RNase Measuring RNA Quantity and Quality Elimination of contaminating yeast genomic DNA in RNA preparations is crucial before cDNA synthesis and PCR. Spectrophotometric readings at 260 and 280 nm were then made for the RNA samples. The yield of RNA varied from 20 to 30 ug from each 5-ml yeast culture and the A260A280 ratios were between 1.8 and 2.3, ours was 2.20 as indicated on the attached sheet data. As a further quality check, 1ug of each RNA sample was subjected to denaturing agarose gel electrophoresis. The presence of 28S and 18S ribosomal RNA over a background smear of mRNA and the absence of both degraded RNA and high molecular weight DNA indicated good quality RNA preparations. Reverse Transcriptase The RNA was reverse transcribed with Superscript III (Invitrogen) using primers that include 2.0 ul of 10ug/ul dT24VN anchored oligo (24mer of dT + any base except T + any base) 0.5ug/ul, 0.5ul of 0.5uglul Random Primers (invitrogen Cat# 48190-011 dilute from the 3ug/ml stock concentration)a capture with 8ul of 5X First- strand buffer (from invitrogen, comes with superscript (III), Dithiothreitol DTT) at a level of 1 mM was added to the wash solutions. The reaction was stop by hydrolyzing the RNA template 10ul per student, pre-aliqoated to make 200ul (20ul of 10N Na0H, 8UL OF 0.5M EDTA, and72 ul of sterile water. Polymerase Chain Reaction Polymerase chain reaction was performed in 50-ul reactions containing ul of each of two primers, 1ul of 10 Mm dNTP mix. The PCR reagents were assembled as a master mix that was split into reactions that received 3.5 X of cDNA from either treated or untreated cells as template. Step 94o C(2mins), step 2 94o C (30secs), step 3 50 degrees, and step 4 72degrees repeated 29cycles and two-cycle intervals thereafter until four samples were taken from each 5ul reaction. We designed primers for the amplification of cDNA from each of three yeast genes. The forward primer MC652- and reverse primer MY653-180base pair (bp) product from HMT1 using an annealing temperature of 50C

Results:

Analysis of the quality of purification techniques indicate that the purified RNA mostly contain high quality RNA with lane 5 leading the top of the list, considering its primer-dimer appearance showing the lowest intensity on the gel. This assumption came from the fact that the efficiency of PCR is related to the quality of the template used. Since the template indicate different sample of RNA purified, we cannot be certain that the smear shown on the gel could be related to different concentration of RNA sample smear-like appearance of RNA samples electrophoresed through a denaturing formaldehyde-agrose gels can results from poor dilution of samples. Comparison of RNA profiles of different students (lane 3) RNA-sample almost degraded, poor quality. (Lane 4) semi-good quality, (land 5) total RNA-sample, great quality- the small amount of carry over between 2.0 and 1.5kb indicate the integrity of the sample. Analysis of first-strand cDNA synthesis after amplication by PCR, see the attached sheet. The product was separated on a 1% agarose gel. Lane, 1 should have contains 100bp molecular weight marker. Lane 2: PCR amplication of the coding region of Hmt1, lane 3. PCR reaction from wild type strain., lane 4 PCR reaction of Hmt1 null strain, and lane 5 6ul of RNA-prep control reaction, Comparing of lane 15 did not show any visible results to be interpreted. I have taken ample precaution to prevent contamination of the electropheric apparatus, expecially the teeth of the comb-while nucleases may be inactive in a presence of a buffer that can denature it, it can be transfer d to and interfere with the later component of an experiment. (RNA Methodologies). I think my error came from mistakenly turning my well upside down while so concern with the accuracy of the amount of buffer to be loaded. However, it has been said that the role of the loading buffer contains glycerol or sucrose, which give it an added density to the sample, making it fall into the well with ease. Thus, my sample might have contained poor quality of the loading buffer, for reasons unknown. Discussion:

These results should have given us information about the gene expression of Hmt1 because mRNA study is an excellent way of studying gene expression, but it is by no means the only one. Because even as we perform these purification techniques and coupled them to PCR for the purpose of studying the gene expression of Hmt1, the mRNA we have isolated, though may give us information about the coding sequence of Hmt1, would give nothing about its translational fate because gene expression is also control at the translational and posttranslational level. Overall we learn about selection of appropriate primers, the quality of template, PCR techniques, RNA manipulation, and the danger, caution involved in the creation of cDNA Moreover, we learn about the selection appropriate controls and our ability to interpret and read electrophoresis results.