TITLE: DNA/RNA extraction and qPCR protocol to assess bacterial abundance in the sponge Halichondria panicea
AUTHORS: Lara Schmittmann, Lucia Pita

[DESCRIPTION]
This protocol summarizes experience and recommendations regarding DNA and RNA extractions from the sponge Halichondria panicea and qPCR to quantify bacterial abundance. 
We have used both bacterial and sponge DNA and RNA for subsequent qPCR. Further, bacterial 16S rRNA amplicon sequencing was performed on DNA and sponge transcriptomes were sequenced successfully from extracted RNA. 

[GUIDELINES]
work under clean bench 
sterilize
contamination

[STEPS]
SECTION: RNA extraction
4. Extract RNA from sponge samples fixed in RNAlater (stored at -80°C)

Clean all surfaces and instruments with ethanol and subsequently RNase-Away
The manufacturers protocol for the RNeasy Mini Kit have been optimized for Halichondria tissue. For more details please refer to the manufacturers protocol
Work clean and quickly to prevent RNA degradation due to long waiting times. We therefore recommend to not process more than 6-12 samples (depending on the experience of the person) at once
 

SECTION: cDNA synthesis
5. cDNA synthesis

Use iScript kit and follow manufacturers guidelines. Only thaw reverse transcriptase directly before adding to master mix and keep on ice!

Use 500 ng RNA per reaction and random primers
SECTION: DNA extraction
1. Extract DNA from samples fixed in RNAlater (stored at -80°C) or flash frozen (stored at -80°C)

Qiagen PowerSoil Kit
Input: 70-200 mg tissue
Process max.24 samples at a time, depending on experience
SECTION: Primers and standard curves
6. Primers


 

 


SECTION: Primers and standard curves
7. Standard curve preparation

Prepare one DNA and one cDNA standard curve. It is recommended to prepare larger volumes in order to use the same standard curve for a complete experiment, or even several experiments
SECTION: DNA extraction
3. Dilute DNA extracts for qPCR

Measure DNA extracts of all samples with qbit

SECTION: Primers and standard curves
7.1. Run 50 µl PCRs with primer pair intended for qPCR with DNA from different sponge individuals, e.g. 8 x 50 µl
SECTION: Primers and standard curves
7.2. Prepare electrophoresis gel with nucleic acid stain (e.g. GelGreen) with large pockets to load 50 µl/well
SECTION: Primers and standard curves
7.3. Cut out gel bands under blue light table with sterile scalpel. Clean up gel bands (Marcherey Nagel Nucleo Spin Clean-up kit) according to the manufacturers protocol and pool PCR products
SECTION: Primers and standard curves
7.4. Prepare dilution series: 
Probably, the concentration of the PCR products is way too high for qPCR. Start by preparing a 1:10 dilution by adding 200 µl PCR product + 1800 µl tRNA water
Then proceed with 1:10 (200+1800 µl) or 1:5 (400+1600 µl) dilutions 

SECTION: Primers and standard curves
7.5. Measure highest standard in QBIT (high sensitivity kit) and calculate concentration for dilutions (~ 1-5 ng/µl)
calculate copy numbers http://cels.uri.edu/gsc/cndna.html 
SECTION: DNA extraction
3.1. Dilute each extract to a concentration of 6 ng/µl with DNase/RNase-free water. Per qPCR reaction we will use 5 µl template, so the total DNA used per qPCR reaction is 30 ng
SECTION: qPCR
8. Recommended plate set-up


 

SECTION: qPCR
9.  
qPCR preparation

Work under the clean bench.
  
Prepare master mix for each primer pair:
     Template      5 µL      gDNA: Total 30 ng per reaction, cDNA: 1:5 dilution of cDNA from 500 ng   DNA        Primer F (50 µM)      0.16 µL      Total 400 nM per reaction        Primer R (50 µM)      0.16 µL      Total 400 nM per reaction        qPCR mix (2x)      10 µL          H2O      4.68 µL          Total      20 µL     
 
SECTION: RNA extraction
4.1. Thaw tissue samples on ice and meanwhile label lysis tubes (label on the side, since during powerlysing step labelling on the lid might disappear!)
SECTION: RNA extraction
4.2. Remove first sample from the cryovial (leave others on ice) and cut on a sterile petri dish with sterile forceps into pieces

SECTION: RNA extraction
4.3. Tare the lysis tube and load with ~80 mg tissue. Place tube on ice and proceed with the next sample (go back to step 4.2). Use a new petri dish and scalpel to prevent cross-contamination.
SECTION: RNA extraction
4.4. Add 600 µl beta-mercaptoethanol/RTL buffer under the hood and disrupt cells in homogenizer for 30 sec at a speed of 3000
SECTION: RNA extraction
4.5. Centrifuge for 10 min at maximum speed
SECTION: RNA extraction
4.6. Remove supernatant carefully without touching the pellet and transfer it to a 2 ml collection tube. From here on, work at RT
SECTION: RNA extraction
4.7. add 1 volume (~370 µl) of 70 % molecular grade ethanol and mix well by pipetting. Proceed immediately to next step
SECTION: RNA extraction
4.8. transfer up to 700 µl including any precipitate to RNeasy Spin column placed in a 2 ml collection tube (provided in kit). Close the lid and centrifuge for 30 sec at 10,000 rpm. Discard flow-through and re-use collection tube. Repeat this step if the sample exceeded 700 µl
SECTION: RNA extraction
4.9. Add 700 µl RW1 buffer and centrifuge for 30 sec at 10,000 rpm. Discard the flow-through
SECTION: RNA extraction
4.10. Add 500 µl RPE buffer and centrifuge for 30 sec at 10,000 rmp. Discard the flow-through
SECTION: RNA extraction
4.11. Add 500 µl RPE buffer and centrifuge for 2 min at 10,000 rpm. Carefully remove the spin column and place it in fresh 2 ml collection tube. Centrifuge for 1 min at max speed to remove any remaining buffer
SECTION: RNA extraction
4.12. Place the spin column in a fresh 1.5 ml Eppendorf tube and add 60 µl EB buffer. Take care to place it directly onto the spin column membrane. Close the lid and incubate for 10 min at RT. Centrifuge for 1 min at 10,000 rpm to elute RNA
SECTION: RNA extraction
4.13. Keep extracts always on ice! Proceed immediately with RNase inhibition and nuclease treatment.
Already preheat incubator or heating block to 37°C (incubator needs much longer to pre-heat than heating block)

 

SECTION: RNA extraction
4.14. Under the clean bench: add 3 µl SUPERase-IN (0.05 volume of RNA) to 60 µl RNA. Mix gently
SECTION: RNA extraction
4.15. Add 6 µl (0.1 volume of RNA) 10x DNAse I buffer and 1.2 µl DNAse and mix gently
SECTION: RNA extraction
4.16. Incubate for 20 min at 37°C, mix frequently
SECTION: RNA extraction
4.17. Add 6 µl resuspended DNAse I inactivation reagent and mix well
SECTION: RNA extraction
4.18. Incubate for 2-5 min at RT, mix every minute by shortly vortexing
SECTION: RNA extraction
4.19. Centrifuge for 1.5 min at 10,300 rpm and transfer supernatant (RNA) to a fresh tube. Take care to not transfer any inactivation reagent (white)
SECTION: RNA extraction
4.20. We recommend to immediately take a 5 µl aliquot and a 2 µl aliquot, and freeze all samples at -80°C. 
Use the 5 µl aliquot to assess quality and quantity (Nanodrop, Qbit, PCR to check contamination with DNA) and the 2 µl aliquot for capillary electrophoresis (e.g. Experion). To avoid thaw-freeze cycles, the main RNA sample can be split in two equal parts, so that one sample can remain frozen until analysis at -80°C if it is intended for e.g. transcriptomics
SECTION: DNA extraction
2. Extract DNA from bacterial recolonization pellets flash frozen (stored at -80°C)

Qiagen Blood+Tissue kit
Input: cell pellet
Process max.24 samples at a time, depending on experience


 

SECTION: DNA extraction
2.1. Follow manufacturers protocol (incubation with proteinase K at 56°C for 30 min)
SECTION: cDNA synthesis
5.2. Store cDNA dilutions at -20°C
SECTION: cDNA synthesis
5.1. Dilute cDNA 1:5 with TE buffer and use this dilution for qPCR
SECTION: qPCR
9.4. 
 Run plate immediately, or store plate at 4°C in the dark and run within 24 h
 
     96°C      2 min               94°C      30 sec      40x        60°C      30 sec (plate read)        72°C      1 min (plate read)        60-90°C      0.5 interval, 5 sec Melt   curve          
 
SECTION: qPCR
9.1. Work in 96-well plate. Pipet 15 µl master mix per well
SECTION: qPCR
9.2. Add 5 µl template per well (use new pipet tip for each well)
SECTION: qPCR
9.3. Seal plate with optical adhesive foil (check which brand fits your plates and qPCR machine) and spin down plate briefly in plate centrifuge
SECTION: qPCR
9.5. Run gel electrophoresis with some qPCR products to check for correct amplification (1 band of correct product length) at least once per experiment
SECTION: Primers and standard curves
7.6.   

SECTION: qPCR
9.6. 

  
SECTION: qPCR
9.7.   


  





SECTION: DNA extraction
1.1. Follow manufacturers protocol (lysis in PowerBeadTubes and PowerLyzer Bead-Based Homogenizer at 3500 rpm, 2x 30 sec bead beating with 45 sec pause in between)
SECTION: DNA extraction
1.2. We recommend to immediately take a 5 µl aliquot for quality and quantity control (Nanodrop, Qbit, PCR to check contamination presence of 16S DNA). DNA extracts can be frozen at -20°C or for long-term storage at -80°C
SECTION: DNA extraction
2.2. We recommend to immediately take a 5 µl aliquot for quality and quantity control (Nanodrop, Qbit, PCR to check contamination presence of 16S DNA). DNA extracts can be frozen at -20°C or for long-term storage at -80°C