To investigate differential geneEPMRNA expression in cell types of a leaf, you a
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To investigate differential geneEPMRNA expression in cell types of a leaf, you analyze mRNA populations in epidermal cells 3ECmRNA EC null mDNA (EC), mesophyll cells (MC), and vascular cells (VC). You isolate mRNA from EC and use7VC MRNA EC null mDNA0006 the mRNA to prepare EC mDNA and EC null mDNA You also isolate mRNA from MC and VC. RNA excess saturation hybridization experiments are done. The complexity of single copy DNA is 5 x 10* bp FRACTION PROBE HYBRIDIZED PROBE EC single copy DNA 0.06 EC EC mDNA MC mRNA MC mRNA VC mRNA EC mDNA EC null mDNA EC mDNA 0.85 0.005 0.85 MC & VC mRNA mixture EC null mDNA 0.008 (9 points) Briefly outline how EC null mDNA is preparedExplanation / Answer
Hybridisation experiments can be performed using this RNA to assess the complexity of any particular gene, or the number of copies that are present in the genome9tandem repetition of that gene).Hybridisation experiments with related sequences or mRNA ,to check the level of divergence among two or more sequences.
Using saturation hybridisation along with probe sequences(excess), The cellular geen should be present in sufficient amount to allow probe hybridisation. Gene number can be estimated by using Cot values (Cot1/2).
STEPS INVOLVED IN PREPARATION OF mDNA FROM MESOPHYLL AND EPIDERMAL CELLS
1.Collection of Mesophyll and Epidermal cells from different plant leaves, cells collected from sections (dissection of the leaf area where the epidermal cells are abundant).
2.Epidermal cells harvested and RNA extraction was performed as prescribed in kit (crude method or kit based RNA extraction (QIAGEN RNA EXTRACTION KIT). Large scale RNA Isolation can be done using trizol based extraction.
3.RNA was eluted using elution buffer
4.RNA concentration was estimated to assess purity and concentration of the RNA in sample using Nanodrop spectrophotometer.
5.Normalisation of the RNA concentration, RNA was diluted further.
6.RNA was subsequently reverse transribed to prepare mDNA by RT-PCR using Reverse transcriptase enzyme (using cDNA synthesis kit). The diluted RNA is used as a template in mDNA reaction mixture
7.mDNA was diluted further 10 times.
8.PCR was performed with control gene (Epidermal cell specific primer, any housekeeping gene primer).
Primer designing of the candidate genes for checking expression.
9.PCR of the candidate genes required to compare the differential gene expression can be performed by optimising the annealing temperature and reaction cycles.
10. Purification of PCR product for sequencing or REAL TIME PCR ANALYSIS.
11) The mutant lacking that particular mRNA OR any knockout created by using Knockout primer specrific for that particular gene of interest. Ones the null mutant created then, same can be performed from those transformed cells.
knock out cell taken---- harvested------- RNA extraction----null mDNA preparation by Reverse transciption-----PCR using gene specific primers.----- Real time PCR---- data analysis to compare.
mRNA Hybridisation with null mDNA fraction of probe hybridised is very less, mDNA is intronless whereas mRNA contains both the exons and introns.
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