RNA processing_Seminar 2_PDF

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Feb 20, 2024

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MCB*2050 Tutorial 2: Quantifying RNA Following qRT-PCR Sample A has a Ct value of 16 and sample B has a Ct value of 11. Fill in the blanks in the following sentence based in this information. Sample A has a (higher/lower) level of (DNA/RNA/protein) in the original sample than Sample B. Diagnosis of Covid-19 by RNA quantification SARS-CoV2, like all coronaviruses it is a single stranded RNA virus meaning that the viral genome consists of a single RNA molecule that is encapsulated by a lipid membrane with a viral protein coat. While the coronavirus genome is the largest of all RNA viruses it still relies on host machinery to perform many functions involved in infection and viral propagation. For example, SARS-CoV2 mRNA is translated by host cell ribosomes to produce all viral proteins. Because this virus infects eukaryotic cells, its mRNA must be recognized by eukaryotic ribosomes. For that reason, coronavirus mRNA shares many of the key features required for eukaryotic translation. Question 1: SARS-CoV2 RNA a) Name 2 key features that we would expect SARS-CoV2 mRNA to contain in order to be translated by mammalian ribosomes and explain why is each one is required. i. ii. b) Examine the following RNA-seq data. In this figure, two cell lines were infected with SARS- CoV2 virus; cells expressing mCherry (blue) and cells expressing the surface receptor ACE2. The ACE2 cells were infected with a low dose of virus (light pink) and high dose of virus (dark red). After 24 hours the total RNA was collected from each cell line and condition RNA sequencing (RNA-seq) was performed and reads were aligned with the SARS-CoV2 genome. This figure provides the number of viral reads (y axis) that aligned with the SARS-CoV2 reference genome and displays where along the genome each read aligned, each tick represents 1,000 base pairs (x axis). i. How many base pairs are in the SARS-CoV2 genome? ii. When comparing cells with ACE2 receptors or mCherry which cells have a higher viral load? (1)

MCB*2050 Tutorial 2: Quantifying RNA iii. Which element of the SARS-CoV2 RNA genome has the most viral reads? Question 2: Develop a Covid-19 diagnostic protocol Because SARS-CoV2 is an RNA virus, protocols for detecting the presence of this virus involve detecting viral RNA. Given the information regarding SARS-CoV2 above and the techniques covered in the Tutorial 2 online material develop a point form protocol for detecting SARS-CoV2 RNA that could be used to diagnose Covid-19. Keep in mind there is more than one way to do this! Work together on a shared document to brainstorm and develop your protocol. Use the space provided below. Consider the following points: i) How will you collect the RNA? ii) Which technique or combination of techniques will you use to detect and analyze viral RNA? • Be sure to include details such as what type of primers, (PolyT, random and/or sequence specific primers) you will use. • What enzymes will be used at the various steps? Hint there are 2 keys enzymes required (regardless of what techniques you use!) iii) Can your method quantify the viral RNA? This is not necessarily required for a diagnostic test however it would be required to detect the level of SARS-CoV2 present on surfaces or to determine an individual’s viral load.

MCB*2050 Tutorial 2: Quantifying RNA Application Assignment Question 3: qPCR Sequence Specific Primer Design (not graded, you do not need to submit this) When designing primers many factors must be considered in order to optimize the PCR amplification and avoid faulty results. Many free primer optimization tools exist online to help one do this but experience has proven that primers need to be rigorously tested with positive and negative controls in order to ensure accurate results for diagnostic tests. The sequence below is the complete SARS-CoV2 N protein, cDNA gene sequence. (Note the N protein is a small structural protein which is routinely used as a target for Covid-19 diagnosis.) 4441 aggaaccttc cttcctgctt gagttcccag atggctggaa ggggtccagc ctcgttggaa 4501 gaggaacagc actggggagt ctttgtggat tctgaggccc tgcccaatga gactctaggg 4561 tccagtggat gccacagccc agcttggccc tttccttcca gatcctgggt actgaaagcc 4621 ttagggaagc tggcctgaga ggggaagcgg ccctaaggga gtgtctaaga acaaaagcga 4681 cccattcaga gactgtccct gaaacctagt actgcccccc atgaggaagg aacagcaatg 4741 gtgtcagtat ccaggctttg tacagagtgc ttttctgttt agtttttact ttttttgttt 4801 tgttttttta aagatgaaat aaagacccag ggggagaatg ggtgttgtat ggggaggcaa 4861 gtgtgggggg tccttctcca cacccacttt gtccatttgc aaatatattt tggaaaacag 4921 ctaaaaaaaa aaaaaaaaaa Use the strategy outlined in the PCR – Primer Design reading from Optional Tutorial: Fundamental Techniques to design sequence specific primers to target this gene. Each primer should be 18-22 nucleotides in length, and create a product no shorter than 100bp, otherwise you can choose the exact location of each. i. Highlight the region on the sequence for which you will design your primers. Remember the reverse primer will be complementary to this sequence while the forward primer will match this sequence as it should bind to the complimentary strand (2 nd strand not shown here). ii. Write out the sequence of your forward and reverse primers in 5’ to 3’. Primers must match the highlighted selections. Forward: 5’- Reverse: 5’- Early diagnostic primers designed to detect SARS-CoV resulted in false positive results. It was determined that poor primer design was to blame as several of their primers sets had a high propensity to form primer dimers. Primer dimers are formed when primers bind to each other and amplify during the qPCR step resulting in false positives. The amplification plot below shows the results from a primer quality control qPCR run in which different primer sets were used in no-template control reactions. Note that a Ct value < 33 is considered positive. iii.) Which primers are problematic? Why?

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