Jul 03, 2023
Version 1
DNA/RNA extraction from fresh-frozen tissue, AllPrep DNA/RNA/miRNA Universal Kit V.1
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Protocol Citation: Annika Fendler 2023. DNA/RNA extraction from fresh-frozen tissue, AllPrep DNA/RNA/miRNA Universal Kit. protocols.io https://dx.doi.org/10.17504/protocols.io.kxygx3mrwg8j/v2Version created by Annika Fendler
License: This is an open access protocol distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited
Protocol status: Working
We use this protocol and it's working
Created: July 03, 2023
Last Modified: July 03, 2023
Protocol Integer ID: 84402
Keywords: DNA, RNA, Fresh-frozen tissue, Qiagen AllPrep, allprep dna, mirna universal kit protocol, rna extraction, mirna universal kit, dna extraction, rna, combined rna, frozen tissue, dna
Abstract
Protocol for combined RNA and DNA extraction from fresh-frozen tissue using the AllPrep DNA/RNA/miRNA Universal Kit.
Materials
AllPrep DNA/RNA/miRNA Universal Kit (50)QiagenCatalog #80224
Genomic DNA ScreenTapeAgilent TechnologiesCatalog #5067-5365
Qubit™ dsDNA BR Assay KitThermo Fisher ScientificCatalog #Q32853
Qubit RNA BR Assay KitThermo Fisher ScientificCatalog #Q10211
RNA ScreenTape and ReagentsAgilent Technologies
EB bufferQiagenCatalog #19086
ß-ME
EtOH
Isoprop
1.5 and 2 ml LoBind tubes
TissueLyser Beads 5 mm
Protocol materials
Genomic DNA ScreenTapeAgilent TechnologiesCatalog #5067-5365
Qubit™ dsDNA BR Assay KitThermo Fisher ScientificCatalog #Q32853
Qubit RNA BR Assay KitThermo Fisher ScientificCatalog #Q10211
RNA ScreenTape and ReagentsAgilent Technologies
EB bufferQiagenCatalog #19086
AllPrep DNA/RNA/miRNA Universal Kit (50)QiagenCatalog #80224
Safety warnings
Dnase I stocks can be used 4 weeks after being thawed but should not be frozen again
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)
Immediately
DNAse I: 70 µl RDD + 10 µl DNase I per sample
Proteinase K: 60 µl AW1 + 20 µl Proteinase K per sample
Tissue preparation
This protocol is for Sample
Optional: Weigh tissue
Enter a complete list of samples used for each experiment below:
| SampleID | Optionel Weigth (mg) | Comment | |
List of tissue sample used in experiment
Transfer tissue in 350 µL for up to 10 mg of tissue 600 µL for 10 to 30 mg or stabilised tissue RLT + ß-ME in 2 ml DNA LoBind tube.
Make sure that the tissue does not defrost.
Add a 5mm bead to each tube and lyse tissue in TissueLyser for 00:02:00 @ 20 Hz
Equipment
TissueLyser II
NAME
Bead Mill
TYPE
QIAGEN
BRAND
85300
SKU
LINK
2m
Spin down for 00:01:00 @ 9000 x g
1m
Turn tube rack and lyse tissue for 00:02:00 @ 20 Hz
2m
Spin down for 00:01:00 @ 9000 x g
1m
Add lysed product to DNA Mini Spin Column
Spin 00:00:30 @ max x g , repeat if any liquid remains on column
30s
Transfer column to a new collection tube and store tube at 4 °C until DNA extraction
Transfer flow-through to new 2 ml LoBind tube
RNA extraction
Add 50 µL for up to 10 mg tissue 80 µL for 10 to 30 mg or stabilised tissue Proteinase K, mix by pipetting
Add 200 µL for up to 10 mg tissue 350 µL for 10 to 30 mg or stabilised tissue EtOH abs., mix by inverting, spin down liquid
Incubate 00:10:00 @ Room temperature
10m
Add 400 µL for up to 10 mg tissue 750 µL for 10 to 30 mg or stabilised tissue EtOH abs. , mix by pipetting
Add 700 µL to RNeasy spin column and spin for 00:00:30 @ max x g and discard flow-through
30s
Repeat until all liquid passed through the column
Add 500 µL RPE
Spin 00:00:30 @ max x g and discard flow-through
30s
Add 80 µL DNase I working solution directly onto the membrane and incubate 00:15:00 at Room temperature
15m
Add500 µL FRN
Spin 00:00:30 @ max x g and don't discard flow-through
30s
Add flow-through again to column
Spin 00:00:30 @ max x g and discard flow-through
30s
Add 500 µL RPE
Spin 00:00:30 @ max x g and discard flow-through
30s
Add 500 µL EtOH abs
Spin 00:02:00 @ max x g and place column in new collection tube
2m
Spin 00:02:00 @ max x g and place column in new 1.5 ml collection tube
2m
Add 30 µL of RNase-free H2O
Incubate 00:01:00 @ Room temperature
1m
Spin down00:01:00 @ 8000 x g
1m
QC: Qubit Qubit RNA BR Assay KitThermo Fisher ScientificCatalog #Q10211 and tape station RNA ScreenTape and ReagentsAgilent Technologies
Expected result
| SampleID | Eluted Volume (µl) | Concentration (ng/ul) | Total mass (ng) | Storage Location | |
Qubit Results RNA
Expected result
Upload PDF from Tapestation here
DNA extraction
11m
350 µL AW1 auf DNA Mini Spin Column geben
Spin 00:00:30 @ max x g and discard flow-through
30s
80 µL Proteinase K working solution directly onto membrane
Incubate 00:05:00 @ Room temperature
5m
Add 350 µL AW1
Spin 00:00:30 @ max x g and discard flow-through
30s
Add 350 µL AW2
Spin 00:02:00 @ max x g and place column in new 2 ml collection tube
2m
Spin 00:01:00 @ max x g and place column in new 1.5 ml collection tube
1m
Add 50 µL EB bufferQiagenCatalog #19086 directly onto membrane
Incubate 00:01:00 @ Room temperature
1m
Spin down 00:01:00 @ 8000 x g
1m
QC: Qubit DNA Qubit™ dsDNA BR Assay KitThermo Fisher ScientificCatalog #Q32853 and tape station Genomic DNA ScreenTapeAgilent TechnologiesCatalog #5067-5365
Expected result
| Sample ID | Eluted Volume (µl) | Concentration (ng/µl) | Total Mass (ng) | Storage Location | |
Results from Qubit DNA
Note
Upload Tapestation results as pdf here