Targeted RNA-Seq
RNA Extraction
Things to check before starting the protocol
Make 1 mg/mL lysozyme in RNase-free 1x TE buffer [prepare 150 µL per RNA sample]
990 uL DEPC treated water
10 uL 100x TE buffer
(1.0 mg, Gram-negative) (3.0 mg, Gram-positive) lysozyme (from chicken egg)
Store on ice
Add 16 µL β-Mercaptoethanol (βME) to 1.6 mL Lysis Solution (Norgen Kit) in an RNase-free eppendorf tube and mix by quick vortexing [prepare 400 µL per RNA sample required]
Make two tubes.
Additional Labeling and preparation
Label 4 RNAse free eppendorf tubes for each biological sample.
Label 1 purification column for each biological sample.
Label 1 eppendorf tube for the loading dye master mix.
Steps to minimize RNAse contamination
Cover hands, arms, head, and body with sterile protective gear (RNAses are in your saliva, your tears, and on your body surface).
Wipe down all bench surfaces with RNAse Away
Use only sterile, disposable plastic tubes, tips, and serological pipettes. It is important to keep a box set of tips for RNA use only -- do not use these tips for any other labwork, e.g. minipreps.
Protocol Steps
Spin down at room temperature 5 mL of bacterial culture at OD600 0.2 (log phase) at max rpm (4,750 rcf), and discard the supernatant [try to withdraw as much supernatant as possible without disturbing the bacterial pellet]
Resuspend the pellet in 100 µL of the lysozyme solution (in TE) by pipetting up and down and/or vortexing and transfer to an RNase-free eppendorf tube. Incubate the resuspension at room temperature for (5 minutes, Gram-negative) (10 minutes, Gram-positive) [if the room temperature is low, incubate at 25 °C]
Add 300 µL of the βME containing Lysis Solution (Buffer RL - Norgen Kit) and mix by vigorous vortexing for 10-15 seconds
Add 200 µL of ethanol (98-100%) to the lysate and mix by vigorous vortexing for 10-15 seconds [use the EtOH bottle from the designated "RNA ONLY" drawer]
Assemble the RNA purification column and the collection tube (Norgen Kit) and add 600 µL of the lysate from Step 4 above to it. Spin at 14,000 rcf for 1 min at room temperature [if all the lysate does not go through the column, spin for another 1 min]
Discard the flow-through and reassemble the column
Add 400 µL of the Wash Solution (Norgen Kit) to the column and spin at 14,000 rcf for 1 min at room temperature [the Wash Solution should have been prepared beforehand by adding RNase-free ethanol to the Norgen supplied bottle]
Discard the flow-through and reassemble the column
Repeat Steps 7 & 8 two more times (total of 3 washes)
Spin the column at 14,000 rcf for 2 min at room temperature in order to dry the column resin
Discard the collection tube and place the column into a fresh RNase-free eppendorf tube [use cold eppendorf tubes]
Add 50 µL of the Elution Solution (Norgen Kit) to the column and spin at room temperature at 200 rcf for 2 min, followed by 14,000 rcf for 1 min [place the RNA on ice-water immediately and keep cold all the time]
For greater recovery, place the column back into the same eppendorf tube and repeat the elution with another 50 µL of the Elution Solution
To the rest of the 95 µL RNA, add 9.5 µL of the 10x TURBO DNase Buffer and 2 µL TURBO DNase (TURBO DNA-free™ Kit) and mix gently by flicking the tube
Incubate at 37 °C for 30 min
Add 10.7 µL DNase Inactivation Reagent (TURBO DNA-free™ Kit) and mix gently by flicking the tube [before pipetting the DNase Inactivation Reagent, flick the tube a couple of times to ensure that the beads are uniformly dispersed]
Incubate at room temperature for 5-6 min while mixing gently by flicking the tube every 2 min
Spin at room temperature at 10,000 rcf for 1.5 min and transfer 100 µL of the supernatant to a fresh RNAse-free eppendorf tube [use cold eppendorf tubes; keep cold all the time]
Take out a 3 µL aliquot in a separate RNase-free eppendorf tube for later analysis [use cold eppendorf tubes; keep cold all the time]
Use the nanodrop to determine RNA concentration using the aliquots taken earlier in Steps 14 and 20 (pre- and post- DNAse treatment)
Run 1 µL each of the RNA samples from Steps 14 & 20 above (post- DNAse treatment) on a 1% agarose gel to check for RNA integrity.
Freeze down all the tubes at -80 °C.
Goal: 100 uL of total RNA at a concentration of 100 - 125 ng/uL. Total RNA extracted 10 -12.5 ug.
RNA Clean-up and Concentration
This protocol uses the Qiagen RNeasy MinElute Cleanup Kit. It can purify up to 45 ug of RNA. Note: This kit does not retain RNA molecules below 200 nucleotides, which comprise 15-20% of Total RNA.
*****Use the two highest concentration tubes.*****
Adjust sample volume to 100 uL. (Norgen Total RNA kit outputs 100uL)
Add 350 uL Buffer RLT and mix well.
Add 250 µl 96%–100% ethanol. Pipette to mix.
Transfer the sample mixture (~700 µl) to an RNeasy MinElute spin column placed in a 2 ml collection tube.
Close the lid, and then centrifuge at 8000 × g for 15 seconds.
Discard the flow-through and collection tube.
Place the RNeasy MinElute spin column in a new 2 ml collection tube.
Add 500 µl Buffer RPE, and then close the lid.
Centrifuge at 8000 × g for 15 seconds.
Discard the flow-through and retain the collection tube.
Add 500 µl 80% ethanol to the RNeasy MinElute spin column, and then close the lid.
Centrifuge at 8000 × g for 2 minutes.
Without allowing the column to contact the flow-through, remove the RNeasy MinElute spin column from the collection tube.
Discard the flow-through and collection tube.
Place the RNeasy MinElute spin column in a new 2 ml collection tube.
Place the spin columns into the centrifuge with at least 1 empty position between columns, which avoids damaging the spin column lids. Orient the lids to point in a direction opposite the rotation of the rotor.
With the lids open, centrifuge at maximum speed for 5 minutes.
Discard the flow-through and collection tube.
Place the RNeasy MinElute spin column in a new 1.5 ml collection tube.
Add 14 uL of RNase-free water to the center of the spin-column membrane
Close the lid, and then centrifuge at maximum speed for 1 minute.
Expect to recover ~12 µl RNA.
Take 2 uL aliquot to determine the concentration and use with analytical gel.
rRNA Depletion
This protocol will use the Illumina Ribo-Zero Removal Kit (MRZMB126). This kit can use a maximum of 5 ug Total RNA and a volume of 26 uL.
Prepare the Beads
Prepare the following consumables:
Item | Storage | Instructions |
Magnetic beads | 2°C to 8°C | Bring to room temperature. Vortex to mix |
Magnetic Bead Resuspension Solution | 2°C to 8°C | Bring to room temperature. |
RNase-free water | 2°C to 8°C | Thaw on ice. Invert to mix, and then centrifuge briefly. |
2. For each reaction, add 225 µl magnetic beads to a 1.5 ml microcentrifuge tube.
3. Place on a magnetic stand, with cap open, and wait until the liquid is clear (~1 minute).
4. Remove and discard all supernatant.
5. Remove from the magnetic stand.
6. Wash 2 times as follows.
Add 225 µl RNase-free water.
Vortex to resuspend.
7. Place on a magnetic stand, with cap open, and wait until the liquid is clear (~1 minute).
8. Remove and discard all supernatant.
9. Remove from the magnetic stand.
10. Add 65 µl Magnetic Bead Resuspension Solution.
11. Vortex to resuspend.
12. Set aside at room temperature.
Hybridize Probes
Prepare the following consumables:
Item | Storage | Instructions |
Ribo-Zero Reaction Buffer | -85°C to -65°C | Thaw on ice. Invert to mix, and then centrifuge briefly. |
Ribo-Zero Removal Solution | -85°C to -65°C | Thaw on ice. Invert to mix, and then centrifuge briefly. |
RNase-free water | 2°C to 8°C | Thaw on ice. Invert to mix, and then centrifuge briefly. |
2. Set a heat block or thermal cycler to 68°C.
3. For each sample, combine the following volumes in a 0.2 ml or 0.5 ml microcentrifuge tube. Pipette to mix.
RNase-free water (26-x µl)
Ribo-Zero Reaction Buffer (4 µl)
RNA sample (x µl)
Ribo-Zero Removal Solution (10 µl)
The total volume per sample is 40 µl.
4. Place on the preheated heat block or thermal cycler and incubate for 10 minutes.
5. Remove from heat, and then centrifuge briefly.
6. Incubate at room temperature for 5 minutes.
Remove rRNA
Set a heat block or thermal cycler to 50°C.
For each sample, do as follows.
Add 40 µl RNA sample to a 1.5 ml tube containing 65 µl washed magnetic beads. Immediately pipette to mix.
Vortex for 10 seconds, and then set aside at room temperature.
3. Incubate at room temperature for 5 minutes.
4. Place on the preheated heat block or thermal cycler and incubate for 5 minutes.
5. Immediately place on a magnetic stand, with cap open, and wait until the liquid is clear (~1 minute).
6. Transfer 85–90 µl supernatant containing depleted RNA to a fresh 1.5 ml tube.
7. Set aside on ice.
Clean Up Depleted RNA
This protocol will use Qiagen RNeasy MinElute Cleanup Kit (74204).
Prepare fresh 80% ethanol from absolute ethanol. Set aside both forms of ethanol on ice.
Adjust the sample to a volume of 100 μl with RNase-free water (Add 10-15 uL water).
Add 350 uL Buffer RLT and mix well.
Add 550 µl 96%–100% ethanol. Pipette to mix.
Add half the sample mixture (~500 µl) to an RNeasy MinElute spin column placed in a 2 ml collection tube.
Close the lid, and then centrifuge at 8000 × g for 15 seconds.
Discard the flow-through.
Add the remaining sample to the spin column.
Repeat steps 6–7.
Discard the flow-through and collection tube.
Place the RNeasy MinElute spin column in a new 2 ml collection tube.
Add 500 µl Buffer RPE, and then close the lid.
Centrifuge at 8000 × g for 15 seconds.
Discard the flow-through and retain the collection tube.
Add 500 µl 80% ethanol to the RNeasy MinElute spin column, and then close the lid.
Centrifuge at 8000 × g for 2 minutes.
Without allowing the column to contact the flow-through, remove the RNeasy MinElute spin column from the collection tube.
Discard the flow-through and collection tube.
Place the RNeasy MinElute spin column in a new 2 ml collection tube.
Place the spin columns into the centrifuge with at least 1 empty position between columns, which avoids damaging the spin column lids. Orient the lids to point in a direction opposite the rotation of the rotor.
With the lids open, centrifuge at maximum speed for 5 minutes.
Discard the flow-through and collection tube.
Place the RNeasy MinElute spin column in a new 1.5 ml collection tube.
Add 14 uL of RNase-free water to the center of the spin-column membrane
Close the lid, and then centrifuge at maximum speed for 1 minute.
Expect to recover ~12 µl RNA.
Take 2 uL aliquot to measure RNA concentration and for gel.
Reverse Transcriptase Reaction and RNase H Degradation
This protocol will use Invitrogen’s SuperScript IV First-Strand Synthesis System
Add the following to a PCR reaction tube.
Component | Volume |
2 μM gene-specific reverse primer | 1 uL |
10 mM dNTP mix (10 mM each) | 1 uL |
Template RNA (10 pg–500 ng mRNA) (up to 11 uL) | 10 uL |
DEPC-treated water (up to 13 uL) | 1 uL |
Pipet mix and briefly centrifuge the components.
Heat the RNA-primer mix at 65 C for 5 min, then put on ice for a least 1 min.
Make the following master mix.
Component | 1 RXN amounts | 3 RXN amounts |
5× SSIV Buffer | 4 uL | 12 uL |
100 mM DTT | 1 uL | 3 uL |
Ribonuclease Inhibitor | 1 uL | 3 uL |
SuperScript™ IV Reverse Transcriptase (200 U/μL) | 1 uL | 3 uL |
5. Pipet mix and briefly centrifuge the components.
6. Add 7 uL of RT reaction master mix to each annealed RNA.
7. Incubate the mix at 50 C for 10 min.
8. Inactivate the reaction by incubating at 80 C for 10 min.
9. Add 1 uL of RNAse H and incubate at 37 C for 20 min.
10. Congrats, you have ss,cDNA!
PCR Amplification
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