NGS rRNA Depletion Kit(rRNA & ITS/ETS) HYD218
This kit uses RNase H digestion to remove ribosomal RNA and 45S ITS/ETS regions from total RNA from humans, mice, and rats to retain messenger RNA (mRNA) and other non-coding RNAs. This kit has good rRNA removal effects for both intact and partially degraded total RNA (such as FFPE RNA). Since degraded FFPE samples usually contain a higher proportion of ITS/ETS than fresh tissue samples, the probes for the 45S ITS/ETS region of humans, mice, and rats in this kit can significantly increase the proportion of valid data in the sequencing results after removal by this kit.
Components
|
Components |
24 T |
96 T |
|
Hybridization Buffer |
72 μL |
288μL |
|
Human Probe Mix (rRNA & ITS/ETS) |
48μL |
192 μL |
|
RNase H Buffer |
72 μL |
288 mL |
|
RNase H |
48 μL |
192 mL |
|
DNase I Buffer |
660 μL |
2×1320 μL |
|
DNase I |
60 μL |
240 μL |
Storage
Store at -25~-15℃ for 1 year.
Instructions
1. Probe hybridization
1. Take out the probe and hybridization buffer from -20°C, thaw, invert and mix, and place on ice for later use.
2. Prepare RNA sample: Calculate the RNA sampling volume based on the input amount and sampleconcentration, and dilute to 10 μL with Nuclease free H2
3. Prepare rRNA removal reaction system in a 200 μL PCR tube according to Table 1.
Table 1 Probe hybridization reaction system
|
Components |
Volume |
|
Hybridization Buffer |
3 μL |
|
Human Probe Mix (rRNA & ITS/ETS) |
2 μL |
|
Total RNA |
10 (100 ng~1 μg) |
|
RNase H |
48 μL |
|
DNase I Buffer |
660 μL |
|
DNase I |
60 μL |
4. Use a pipette to gently blow and mix, and centrifuge the reaction solution to the bottom of the tube.
5. Place the above PCR tube in the PCR instrument and perform the probe hybridization reaction according to the reaction program shown in Table 2.
Table 2 Probe hybridization reaction program
|
Temperature |
Time |
|
Heat lid to 105°C |
On |
|
95°C |
2 min |
|
95°C-22°C |
0.1°C/s |
|
22°C |
5 min |
|
4°C |
hold |
2. RNase H digestion
1. Take out the RNase H digestion reagent from -20°C, thaw it, invert and mix it, and place it on ice for later use. Prepare the RNase H digestion reaction system as shown in Table 3.
Table 3 RNase H digestion reaction system
|
Components |
Volume |
|
RNase H Buffer |
3 μL |
|
RNase H |
2 μL |
|
Previous step product |
15 μL |
Note: RNase H Buffer and RNase H need to be added separately. If you need to prepare a mix due to a large amount of samples, please prepare it immediately before use, otherwise it will affect the removal effect.
2. Use a pipette to gently blow and mix, and centrifuge the reaction solution to the bottom of the tube.
3. Place the above PCR tube in the PCR instrument and set the reaction program: hot cover 50°C; 37°C, 30 min;4 °C, hold, and perform RNase H digestion reaction.
3. DNase I digestion
1. Take out the DNase I digestion reagent from -20°C, thaw it, mix it by inversion, and place it on ice for later use. Prepare the DNase I digestion reaction system as shown in Table 4.
Table 4 DNase I digestion reaction system
|
Components |
Volume |
|
DNase I Buffer |
27.5 μL |
|
DNase I |
2.5 μL |
|
Previous step product |
20 μL |
2. Use a pipette to gently pipette to mix, and centrifuge the reaction solution to the bottom of the tube.
3. Place the above PCR tube in a PCR instrument and set the reaction program: hot cover 50°C; 37°C, 30 min; 4°C, hold, and perform DNase I digestion reaction.
4. RNA purification
1. Preparation: Take the NGS RNA Cleaner magnetic beads out of the refrigerator andequilibrate at room temperature for at least 30 min. Prepare 80% ethanol with Nuclease free H2
2. Vortex or invert the magnetic beads to ensure thorough mixing.
3. Pipette 110 μL NGSRNA Cleaner (2.2×, Beads:DNA=2.2:1) into the product of the previous step, pipette to mix thoroughly, and incubate at room temperature for 5 min.
4. Place the PCR tube in a magnetic rack to separate the magnetic beads and liquid. After the solution is clear(about 3 min), carefully remove the supernatant.
5. Keep the PCR tube in the magnetic rack, add 200 μL freshly prepared 80% ethanol to wash the magnetic Incubate at room temperature for 30 sec, and carefully remove the supernatant.
6. Repeat step 5)for a total of two rinses. Use a 10 μL pipette to aspirate the remaining liquid.
7. Keep the PCR tube in the magnetic rack and open the lid to dry the magnetic beads at room temperature (5~10min).
8. RNA elution: Remove the PCR tube from the magnetic rack, add 11 μL Nuclease free H2O(or use thecorresponding volume of Nuclease free H2O or elution buffer suitable for downstream experiments), use a pipette to gently blow until fully mixed, and let it stand at room temperature for 5 min.
9. Centrifuge the PCR tube briefly and place it on a magnetic stand. After the solution isclear (about 3 min), carefully transfer 10 μL of supernatant (adjustable according to the actual elution volume selected in step 7) to a new Nuclease free PCR tube.
Note: Please perform downstream experiments immediately on the eluted samples, or store them at -80 °C.
Notes
1. Please use RNase-free consumables and clean the experimental area regularly. It isrecommended to use Thermo Fisher's RNAZapTM high-efficiency nucleic acid removal spray to remove RNase contamination.
2. RNA samples should not contain genomic DNA contamination. If there is gDNA residue in the sample, it should be digested and purified by DNase I before using this kit.
3. The maximum input volume of RNA sample is 10 μL. If the sample volume is large, it can be concentrated first.
4. If the RNase H digestion step needs to be used with Mix, please prepare it now; there will be surplus in the 24 T specifications of this component.
5. For your safety and health, please wear a lab coat and disposable gloves when operating.
6. This product is for scientific research purposes only!