DNA/RNA extraction from fresh-frozen tissue, AllPrep DNA/RNA/miRNA Universal Kit
Annika Fendler
Abstract
Protocol for combined RNA and DNA extraction from fresh-frozen tissue using the AllPrep DNA/RNA/miRNA Universal Kit.
Before start
Preparations:
FRN buffer: Add 42 ml Isoprop to new bottle
RPE buffer: Add 44 ml EtOH to new bottle
AW1 buffer: Add 25 ml EtOH to new bottle
AW2 buffer: Add 30 ml EtOH
DNAse I stocks: 550 µl RNase-free water to lyophilised DNAse I, aliquot and store at -20°C for 9 months)
Steps
Prepare Working Solutions
From
DNAse I working solution: 70µL RDD + 10µL DNase I working solution per sample
Included within
Proteinase K working solution for DNA isolation: 60µL AW1 + 20µL Proteinase K per sample
Included within
Add 10µL ß-Mercaptoethanol (not included in kit) per 1mL of RLT plus buffer
Tissue preparation
This protocol is for Sample
Optional: Weigh tissue before start to decide for volume
Enter a complete list of samples used for each experiment below:
| A | B | C | D | E |
|---|---|---|---|---|
List of tissue sample used in experiment
Keep tubes with tissue on dry ice and make sure that they do not thaw during processing.
Transfer the tissue piece into a 6-well plate or small petry dish placed on dry ice and cut an appropriate piece of tissue (ca. 10-30 mg) with a safety scalpel.
Transfer tissue in 350µL 600µL RLT + ß-ME in 2 ml DNA LoBind tube.
Add a 5mm bead to each tube and lyse tissue in TissueLyser for 0h 2m 0s @ 20 Hz
Equipment
| Value | Label |
|---|---|
| TissueLyser II | NAME |
| Bead Mill | TYPE |
| QIAGEN | BRAND |
| 85300 | SKU |
Spin down for 0h 1m 0s @ 9000x g,0h 0m 0s
Turn tube rack and lyse tissue for 0h 2m 0s @ 20 Hz
Spin down for 0h 1m 0s @ 9000x g,0h 0m 0s
Add lysed product to DNA Mini Spin Column
Spin 0h 0m 30s @ maxx g,0h 0m 0s , repeat if any liquid remains on column
Transfer column to a new collection tube and store tube at 4°Cuntil DNA extraction
Transfer flow-through to new 2 ml LoBind tube
RNA extraction
Add 50µL 80µL Proteinase K (not diluted), mix by pipetting
Add 200µL 350µL EtOH abs., mix by inverting, spin down liquid
Incubate 0h 10m 0s @ Room temperature
Add 400µL 750µL EtOH abs. , mix by pipetting
Add 700µL to RNeasy spin column and spin for 0h 0m 30s @ maxx g,0h 0m 0s and discard flow-through
Repeat until all liquid passed through the column
Add 500µL RPE
Spin 0h 0m 30s @ maxx g,0h 0m 0s and discard flow-through
Add 80µL DNase I working solution directly onto the membrane and incubate 0h 15m 0s at Room temperature
Add500µL FRN
Spin 0h 0m 30s @ maxx g,0h 0m 0s and don't discard flow-through and place column in new collection tube
Add flow-through again to column
Spin 0h 0m 30s @ maxx g,0h 0m 0s and discard flow-through
Add 500µL RPE
Spin 0h 0m 30s @ maxx g,0h 0m 0s and discard flow-through
Add 500µLEtOH abs
Spin 0h 2m 0s @ maxx g,0h 0m 0s and place column in new collection tube
Spin 0h 2m 0s @ maxx g,0h 0m 0s and place column in new 1.5 ml collection tube
Add 30µLof RNase-free H2O
Incubate 0h 1m 0s @ Room temperature
Spin down0h 1m 0s @ 8000x g,0h 0m 0s
QC: Qubit
DNA extraction
350µL AW1 auf DNA Mini Spin Column geben
Spin 0h 0m 30s @ maxx g,0h 0m 0s and discard flow-through
80µLProteinase K working solution directly onto membrane
Incubate 0h 5m 0s @ Room temperature
Add 350µL AW1
Spin 0h 0m 30s @ maxx g,0h 0m 0s and discard flow-through
Add 350µL AW2
Spin 0h 2m 0s @ maxx g,0h 0m 0s and place column in new 2 ml collection tube
Spin 0h 1m 0s @ maxx g,0h 0m 0s and place column in new 1.5 ml collection tube
Add 50µL
Incubate 0h 1m 0s @ Room temperature
Spin down 0h 1m 0s @ 8000x g,0h 0m 0s
QC: Qubit DNA
