RNA Detection

Chapter 2 Quantification of 2 0 O-Me Residues in RNA Using Next-Generation Sequencing (Illumina RiboMethSeq Protocol) Lilia Ay ...

transcriptome, this task is unimaginable without appropriate high-throughput analysis techniques. Next-generation sequencing app ...

upstream T7 RNA polymerase promoter (underlined in the sequence) to perform T7 transcription. 18S forward primer: 50 - TAATACGAC ...

80% ethanol. Mini Quick Spin RNA column (e.g., Roche). UV spectrophotometer (e.g., NanoDrop 2000c). 2.3 Yeast Total RNA Extrac ...

Chloroform. 3 M NaOAc in water, pH 5.2. 96% ethanol. 80% ethanol. Refrigerated tabletop centrifuge. 2.5 Yeast rRNA Reconstruct ...

0.2 mL RNase-free PCR tubes, strips of 8. Flat PCR Caps, strips of 8. PCR thermal cycler. 2.8 RNA Purification 1. RNeasy MinEl ...

Perform PCR on a thermal cycler using the following program parameters: 95C for 5 min; 25 cycles [denaturation 95C for 30 s, ...

Check the quality of the transcripts by using the Agilent 2100 Bioanalyzer (seeNote 5)(seeSubheading3.4) and proceed to yeast r ...

3.4 Purification of 18S and 25S rRNA from Yeast Total RNA The protocol for RNA isolation is derived from [13]. The aim of this t ...

Cool down the gel at room temperature and poor it to a gel chamber with appropriate combs until complete solidification. When t ...

Dilute your RNA mixes to 3–5 ng/μL with RNase-free water to be within the optimal range concentration of the assay (Pico RNA ch ...

Report each area for 18S and 25S rRNA indicated for each lane under the electropherogram and compare them between the lanes. On ...

3.6.2 RNA Fragmentation Quality Control Prepare your samples by mixing 5μL of RNA marker (green cap vial) with 1μL of your frag ...

3.9 Library Preparation Using NEBNext®Multiplex Small RNA Library Prep Set for Illumina® Mix 6μL of RNA sample with 1μLof3^0 SR ...

Add 700μL of wash buffer to the column and centrifuge at full speed for 30 s. Discard the flow-through. Centrifuge the empty co ...

Add 1μL of each of your library to 11 different tubes of 1.5 mL already containing 5μL of RNA marker (green cap vial). Mix by p ...

4 Notes Plasmid pHW18 was a generous gift from Dr. D. Tollervey. It was used as a template to amplify yeast 18S and 25S rDNA. T ...

Incubation may be further continued for 4–6 h depending on transcription efficiency. The quality of the RNA transcripts may be ...

The rRNA ratio 25S/18S calculated for yeast total RNA prep- aration used as a control should be equivalent to 25S and 18S rRNA ...

References Liu N, Pan T (2016) N6-methyladenosine- encoded epitranscriptomics. Nat Struct Mol Biol 23(2):98–102. doi:10.1038/ns ...