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==Experimental Outline== #Remove DNA from RNA (UHRR 740000-41) #*Shouldn't be completely necessary #Biotinylate RNA #Attach RNA to magnetic streptavidin beads #V6 Padlock Probe capture to RNA #Quantify captured products by qPCR #*Also adds Illumina sequencing adapters ===Sample Groups=== *Use Universal Human Reference RNA (UHRR 740000-41) *3 RNA template concentrations for each (30ng, 150ng and 820ng) **For DNA template positive control only do 1 sample using 300ng gDNA *Sample 1: gDNA template captured by V6 padlock probes with Ampligase at 55C **Standard padlock probe capture except keep at 55C whole time *Sample 2: RNA template captured by V6 padlock probes with SplintR at 25C **Should be ligated but non-specific because low temp *Sample 3: biotinylated and streptavidin bead linked RNA captured by V6 padlock probes with SplintR at 25C **No washing or pull down **Should be same as Sample 2 unless RNA is lost during biotin or streptavidin bead steps *Sample 4: RNA template captured by V6 with Ampligase at 55C **Negative control to see difference between SplintR and Ampligase *Sample 5: No template with V6 and SplintR at 25C **NTC checks for ssDNA ligase activity *Sample 6: biotinylated and strept bead linked RNA captured by V4 padlock probes at 55C followed by wash and then SplintR & gDNA at 25C **V4 padlock probes should be targeting wrong strand and completely washed away before *Sample 7: biotinylated and strept bead linked RNA captured by V6 at 55C followed by wash and then SplintR at 25C *Summary: **3 samples with biotinylated RNA **2 samples where RNA is pulled down by magnetic beads and washed to remove non-specific binding
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