Aug 02, 2023
Version 3
DNA/RNA extraction from fresh-frozen tissue, AllPrep DNA/RNA/miRNA Universal Kit V.3
- 1Charite
- Annika Fendler: Creator
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/v3Version 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: August 02, 2023
Last Modified: August 02, 2023
Protocol Integer ID: 85855
Keywords: DNA, RNA, Fresh-frozen tissue, Qiagen AllPrep
Abstract
Protocol for combined RNA and DNA extraction from fresh-frozen tissue using the AllPrep DNA/RNA/miRNA Universal Kit.
Guidelines
DNase I stocks can be used 4 weeks after being thawed but should not be frozen again.
Never thaw tissue when processing.
Always use a fresh scalpel and plate when cutting tissues to prevent cross contamination.
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
RNase-Free DNase SetQiagenCatalog #79254
Step 1
AllPrep DNA/RNA/miRNA Universal Kit (50)QiagenCatalog #80224
In Materials and 2 steps
Genomic DNA ScreenTapeAgilent TechnologiesCatalog #5067-5365
Materials, Step 49
Qubit™ dsDNA BR Assay KitThermo Fisher ScientificCatalog #Q32853
Materials, Step 49
Qubit RNA BR Assay KitThermo Fisher ScientificCatalog #Q10211
Materials, Step 36
RNA ScreenTape and ReagentsAgilent Technologies
Materials, Step 36
EB bufferQiagenCatalog #19086
Materials, Step 46
Safety warnings
All steps with ß-ME should be done under the hood.
Reduce use as sharps when working with primary tissue and notify Bettina Ergün in case of an accident.
Ethics statement
Primary patient tissue can only be used after appropriate ethics approval has been obtained. Only use tissue for which an EVE has been signed and which has been approved to be used for the study you are working on.
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)
Prepare Working Solutions
Prepare Working Solutions
From RNase-Free DNase SetQiagenCatalog #79254
DNAse I working solution: 70 µL RDD + 10 µL DNase I working solution per sample
Included within AllPrep DNA/RNA/miRNA Universal Kit (50)QiagenCatalog #80224
Proteinase K working solution for DNA isolation: 60 µL AW1 + 20 µL Proteinase K per sample
Included within AllPrep DNA/RNA/miRNA Universal Kit (50)QiagenCatalog #80224
Add 10 µL ß-Mercaptoethanol (not included in kit) per 1 mL of RLT plus buffer
Tissue preparation
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:
| SampleID | Type (TURB, T1, T2) | Optional: Weight (mg) | Comment | DNA (ng/ul) | |
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 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.
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
RNA extraction
Add 50 µL for up to 10 mg tissue 80 µL for 10 to 30 mg or stabilised tissue Proteinase K (not diluted), 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 and place column in new collection tube
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
Note
Upload results from Tapestation here
DNA extraction
DNA extraction
11m
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
Note
Upload Tapestation results as pdf here.