Bioch611_8_28_2007.ppt.ppt

RT-PCR Basic Reaction Mixture: RNA dNTPs Primers 1x Buffer Reverse Transcriptase RNase Inhibitor Thermophilic Polymerase PCR based cDNA Cloning Commonly used to make a cDNA library from mRNA Northern Blots Similar to southern blots in that it involves the separation of RNA species on agarose gels and their transfer to nitrocellulose. Unlike Southern blots, Northern blots are separated on a denaturing formaldehyde-agarose gel and gels are not treated with NaOH prior to transferring to nitrocellulose. Nuclease Protection Assay (NPA) The basis method involves: Hybridize in solution a single-stranded antisense probe(s) to an RNA sample After hybridization, any unhybridized probe and sample RNA are removed by digestion with nucleases The nucleases are inactivated and the remaining probe:target hybrids are precipitated. These products are separated on a denaturing polyacrylamide gel and are visualized Nuclease protection assays (NPAs) include both ribonuclease protection assays (RPAs) and S1 nuclease assays These two assays are an extremely sensitive method for the detection, quantification and mapping of specific RNAs in a complex mixture of total cellular RNA. There are several advantages to this technique including (1) multiple mRNAs can be assayed in a single RNA preparation (2) the length of each gene fragment is unique allowing multiple probes to be synthesized together and hybridized to a single target sample (3) highly specific and sensitive assay allowing the detection of sub-picograms quantities of specific mRNA Detailed Information can be found at: /techlib/basics/npa/ RNase Protection Assay What in the world would you use this for?? Example: You knockout a transcription factor in a mouse. You want to know if the lack of this transcription factor affects the transcription of gene X, gene Y, and gene Z. You can probe for the presence of the mRNA for each of the genes in question using RNase Protection Assays Primer Extension Primer extension is use