RNA Extraction Protocol for Shorea
mpfsum
Abstract
RNA extraction protocol using CTAB method optimized for leaf and bud samples from Shorea curtisii.
Adapted from extraction protocol for Shorea beccariana (see attached publication).
Steps
Lysis
Weigh BUD 40mg or LEAF 60mg and transfer into a 1.5 mL tube.
Ground in CTAB buffer (3% CTAB, 1.4 M NaCl, 20 mM EDTA, 100 mM Tris–HCl pH 8.0, 0.2% b-mercaptoethanol) using tissuelyzer.
Incubate at 60°C for 0h 45m 0s.
Precipitation
Add 1 volume of chloroform.
Mix by vortex but not too vigorous.
Ensure that the two phases of the mixture are homogenized well.
Centrifuge at5000rpm .
Transfer the supernatant into a new 1.5 mL tube.
Add 2/3 volume of isopropanol.
Mix gently (turning the tubes upside down 0h 2m 0s ).
Centrifuge 5000rpm
Discard solution carefully.
Washing
Add 700µL ethanol.
Mix gently (flicking the tube) until the pellet no longer stick to the tube.
Centrifuge 5000rpm.
Discard solution carefully.
Air dry the pellet 0h 1m 0s.
Repeat Washing if necessary.
Resuspension
Resuspend the pellet in 50µL RNAse-free water.
Flick the tube until the pellet is no longer visible.
If the pellet is difficult to dissolve, leave it for ~30 min before flicking again.
If it still does not dissolve, add more RNAse-free water AND adjust the subsequent steps accordingly.
Genomic DNA Digestion
Removed gDNA using
Follow the manufacturer's protocol.
Purification
Purification is done using
Add 350µL RLT buffer.
Mix gently (flicking).
Add 250µL absolute ethanol.
Mix gently (flicking).
Transfer the solution into RNeasy Mini Spin column.
Centrifuge 10000rpm.
Discard the flow through.
Add 700µL RW1 buffer to the column.
Centrifuge 10000rpm.
Discard the flow through.
Add 500µL RPE buffer to the column.
Centrifuge 10000rpm.
Discard the flow through.
Add 500µL RPE buffer to the column.
Centrifuge 10000rpm.
Discard the flow through.
Replace the collection tube.
Centrifuge 14000rpm to further dry the membrane.
Replace the collection tube with a new 1.5 mL tube.
Add 50µL RNAse-free water directly onto the membrane.
Incubate at Room temperature for 0h 5m 0s.
Centrifuge 10000rpm.
Store the extracted RNA in -20°C or -80°C for longer storage.
Quality Assessment
Evaluate the RNA quality using Agilent 2100 Bioanalyzer.
