Rna Discussion- The mRNA Differential Display Assays

First, As the chief technical officer at a large pharmaceutical company, you've been called into the research and development (R&D) department because one of the teams is having terrible difficulties with their mRNA differential display experiments. It quickly becomes evident that the lab techs are inexperienced and really don't know why they are getting nothing but smears, or nothing at all, when they run their gels (perform electrophoresis). Your task is to produce a troubleshooting checklist to help resolve these problems.
Develop a list of 5-10 things that you would check, in the order in which you would check them, to help improve the outcome of the mRNA differential display assays. You should create a bulleted or numbered list, with a very brief explanation of why that item is on your list.
Second. You are free to suggest changes to the following three posts’ troubleshooting checklists which, you believe, would help get to the root of the problem, and be sure to explain your suggested changes. Your responses should be well thought out, consist of at least several sentences, be courteous, and be respectful. Be sure to respond to then following three postings.

First, As the chief technical officer at a large pharmaceutical company, you've been called into the research and development (R&D) department because one of the teams is having terrible difficulties with their mRNA differential display experiments. It quickly becomes evident that the lab techs are inexperienced and really don't know why they are getting nothing but smears, or nothing at all, when they run their gels (perform electrophoresis). Your task is to produce a troubleshooting checklist to help resolve these problems. Develop a list of 5-10 things that you would check, in the order in which you would check them, to help improve the outcome of the mRNA differential display assays. You should create a bulleted or numbered list, with a very brief explanation of why that item is on your list. Second. You are free to suggest changes to the following three posts’ troubleshooting checklists which, you believe, would help get to the root of the problem, and be sure to explain your suggested changes. Your responses should be well thought out, consist of at least several sentences, be courteous, and be respectful. Be sure to respond to then following three postings. First post Troubleshooting mRNA Differential Display Experiments Step 1: I would check to see if the RNA has been isolated correctly ◦ Has your RNA been correctly Isolated using the correct Lysis Buffer? ▪ For your specific sample (Tissue or cell culture) you will need to choose between using Harsh or Gentle lysis buffers I would check this step to ensure that enough RNA has been collected and has been collected correctly. *Step 2: Make sure that a no-reverse transcriptase control has been run ◦ This step should be checked because running this control will ensure that product is being generated. This is necessary to conduct the mRNA differential display experiment. Without any product there would be no experiment. *Step 3: Has a downstream primer been designed? Checking this step will allow the investigator to determine if the fault lies within not using a downstream primer. Downstream primers need to be used to allow all possible mRNAs to be reverse transcribed. Also, these primers create subpopulations, and subpopulations will get rid of the smearing issue. To do this: ◦ Choose between 3’ nucleotide anchor or 3’ dinucleotide anchor    Step 4: Check how the cDNA was synthesized ◦ You want to be sure that your cDNA sample has been synthesized appropriately ▪ Have you synthesized the longest cDNAs? ▪ The use of the MMLV reverse transcriptase can help with this ▪ Have the appropriate template, RNA, and primer (oligo(dT) been used? Step 5: Check the design of the Upstream Primers ◦ I would do this to ensure that your cDNA has this anchor attached to allow for PCR amplification. Also, in this step I would make sure that the upstream primer combinations have not been all run simultaneously. *Step 6: Make sure identical primer combinations were loaded correctly into the gel I would check this step because in order to get a side by side comparison, each reaction pair must be placed in the correct order. This would produce better results when looking at the gel. For Example: ◦ Downstream Primer should be the same for all “State” reactions ◦ Upstream Primer should be identical for the “State” reactions coupled together; for example: State A and State B loaded into lane 1 and 2 should have the same Upstream Primer, while State A and State B loaded into lanes 3 and 4 should have the same Upstream Primer, and so on. Second post Troubleshooting mRNA DD It is important to ensure that major steps in the mRNA differential display are performed correctly. Proceed from the beginning: Step 1:Isolate the RNA by identifying the modulated sequences. Isolate highquality RNA with guanidinium acid phenol ◦ Ensure that all contaminated gDNA is removed to ensure exceptional performance. Step 2:For each high-quality RNA sample, perform numerous cDNA synthesis reactions ◦ Reverse transcribe only a small amount of cDNA in a reaction tube. Step 3:The next step is to synthesize the first-strand cDNA. ◦ This synthesis is typically performed using 35S or 32 Ensure that the first-strand cDNA is stored properly so that long-term RNA can be used as well. synthesize ◦ Synthesize the longest cDNAs Step 4: Perform PCR amplification using upstream and downstream primers. ◦ It is important to note that upstream primers(10-mers or 13-mers) are less structured than downstream primers. About seven or eight of the bases of upstream primer are expected ◦ store some of the first-strand cDNA synthesis reactions at –80 After completing these steps, the user should be successful. Please consider the following steps if the user continues to face complications: Step 5: When RNA is being reverse transcribed, ALWAYS run a no-reverse transcriptase, before completing the next task, treat the sample with DNase, if PCR product is generated in an RT− control reaction. Step 6: Run with only ONE primer, this ensures that the primers are not self-priming. Step 7: Assay two concentrations of the SAME RNA sample. ◦ Recommended step: a pilot study, but not necessary Step 8:Confirm the expression is correct. Run a second time. We apologize for the inconvenience and hope that your company overcomes the technical difficulties that have arisen. If your company has any further questions, please contact us anytime. Thank you. Third post If you are in need of improving the outcome of the mRNA differential display assays, please follow this checklist provided below. Step 1: Isolating RNA ◦ Make sure to use the provided guanidinium-containing reagents ◦ Try using guanidinium-acid-phenol for isolation and purification methods ◦ Make sure all genomic DNA is removed (DNase-treating) ◦ Validate the removal of DNA by the no-reverse transcriptase control reaction Step 2: cDNA downstream primer design ◦ Check to make sure your downstream primer is allowing mRNAs to be reverse transcribed ◦ This will generate subpopulations to reduce smearing in the bands ◦ Try using downstream primers with 3′ dinucleotide anchors (VV) to decrease the chance of false positives Step 3: cDNA synthesis ◦ Check to make sure the first-strand of cDNA is stored appropriately so that longterm mRNA of unique samples does not become an issue ◦ Check to see if you synthesized the longest cDNA so that it is long enough to base-pair with the upstream primers Step 4: PCR amplification ◦ Upstream primers should be less structured than that of the downstream primers ◦ Upstream primers should be 10-mers or 13-mers ◦ Try running the possible primer combinations individually ◦ Try to store some of the first-strand cDNA synthesis reactions at -80 Additional guidance if problem is not resolved: ◦ Run a non-reverse transcriptase ◦ Run control reactions with one primer ◦ Assay two concentrations of the same RNA sample ◦ Develop a pilot set of reactions before ◦ Eliminate false positives with a second run using only those primer pairs that show differences in the first run