Question

B. Based on lanes 4 and 3, which cell type has the more tubulin protein-NIH/3T3 cells? Co cells? About the same? Can you tell? 14. As a way of contrasting the results of various molecular methods, answer each of the following questions. Note that each of these questions asks you to think about two methods that might seem superficially to yield similar information, but to focus how the results are different. A) What information would you get from a northern blot that you would not get from reverse transcriptase-PCR if used to detect the same mRNA B) What information would you get from a Southern blot that you would not get from a PCR experiment if used to detect the same genetic locus in human genomic DNA? C) Given your answers to A and B, why is it more common to choose to use the PCR based methods instead of the blot-based methods? D) What information would you get from a western blot that you would not get from a northern blot when used to analyze the expression of the same gene?

Answer 1

A) Northern blotting is a cumbersome procedure used to study mRNA. The RNA sample is first separated by size using electrophoresis and then transfered onto the membrane crosslinked and hybridised by the labelled probe. The membrane is then washed extensively to remove background noise and then subjected to exposure . The major drawback of northern blotting was the interference of RNAse that degraded the sample through contamination. However nowadays RNase free reagents are being used to get clear results. Whereas reverse transcriptase PCR requires fewer methods and execution to stressfree. This method can be used to detect low levels of mRNA which northern blotting cannot acheive. The RNA template is copied into complementary DNA/cDNA by retroviral reverse transcriptase.This cDNA is then amplified several times through PCR. The only advantage of northern blotting over RTPCR is that northern blotting gives information about the transcript size, identifies alternatively spliced transcripts and multigene family while RT-PCR just detects and quantifies the mRNA.

B) Southern blotting is a method used for detecting a particular DNA fragment or a gene in the sample.It can identify mutations, deletions and gene arrangements.It can detect multiple homologus genes ina genome whereas PCR just amplifies the specific DNA fragment. In case of working with homologous genes, if two bands are observed through PCR it is most likely to be non-specific and contamination of the sample.

C) PCR is the go to method for various experiments over blots because

• PCR is less expensive and faster.
• PCR is highly sensitive and quantitative while blots are qualitative.
• Much less DNA is required for any analysis through PCR when compared to the blots.

D) Northern blotting is the technique used to detect the mRNA levels while analyzing the gene by using labelled RNA probes whereas western blotting analyzes gene expression by directly measuring the protein levels using primary antibodies as probes.