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Hieff NGS® MaxUp rRNA Depletion Kit (Human/Mouse/Rat)

Product SKU

Specifications

Price

12253ES24

24 rxn

$1754.45

12253ES96

96 rxn

$5768.85

Introduction

Hieff NGS® MaxUp rRNA Depletion Kit (Human/Mouse/Rat) is designed to deplete rRNA from human, mouse, and rat total RNA based on RNAse H-based workflow, while leaving mRNA and other non-coding RNA. This kit is suitable for both intact and degraded RNA samples (i.e. FFPE RNA).

Application

The resulting rRNA-depleted RNA is suitable for RNA-Seq, random-primed cDNA synthesis, or other downstream RNA analysis.

Contents

Components

12253ES08

12253ES24

12253ES96

12253-A Hybridization Buffer

24 μL

72 μL

288 μL

12253-B Probe Mix(H/M/R)

8 μL

24 μL

96 μL

12253-C RNase H Buffer

24 μL

72 μL

288 μL

12253-D RNase H

16 μL

48 μL

192 μL

12253-E DNase I Buffer

220 μL

660 μL

2×1320 μL

12253-F DNase I

20 μL

60 μL

240 μL

Storage

All the components can be stored at -20℃ for one year

Required Materials Not Included

1. RNA clean beads:Hieff NGS® Cleaner(Cat#12602)or equivalent.

2. Other materials: Ethanol (freshly prepared)、ddH2O、Pipettes、PCR tubes、Magnetic rack、Thermocycler.

Protocols

1.1 Dilute 10 ng–1 μg of total RNA with Nuclease-free Water to a final volume of 11 μl in a PCR tube. Keep the RNA on ice.

1.2 Assemble the following RNA/Probe hybridization reaction on ice according to Table 1.

Table 1 RNA/Probe hybridization reaction

Components

volume(μL)

Hybridization Buffer

3

Probe Mix(H/M/R)

1

Total RNA

11(100 ng~1 μg)

Total

15

1.3 Mix thoroughly by gently pipetting up and down at least 10 times. Briefly spin down the tube in a microcentrifuge to collect the liquid from the side of the tube.

1.4 Place tube in a thermocycler and run the following program with the heated lid set to 105°C.

Table 2 Reaction program of RNA/Probe hybridization

Temperature

Time

Hot lid 105°C

On

95℃

2 min

95℃-22℃

0.1℃/s

22℃

5 min

4℃

hold

2. RNase H Digestion

2.1 Assemble the following RNase H digestion reaction on ice according to Table 3.

Table 3 RNase H digestion reaction

Components

Volume(μL)

RNase H Buffer

3

RNase H

2

Hybridized RNA (Step 1.4)

15

Total

20

2.2 Mix thoroughly by gently pipetting up and down at least 10 times. Briefly spin down the tube in a microcentrifuge to collect the liquid from the side of the tube.

2.3 Place tube in a thermocycler and run the following program: lid 50℃; 37℃, 30 min, 4℃, hold.

3. DNase I Digestion

3.1 Assemble the following DNase I digestion reaction on ice according to Table 4.

Table 4 DNase Ⅰ digestion reaction

Components

Volume(μL)

DNase I Buffer

27.5

DNase I

2.5

RNase H treated RNA (Step 2.3)

20

Total

50

3.2 Mix thoroughly by gently pipetting up and down at least 10 times. Briefly spin down the tube in a microcentrifuge to collect the liquid from the side of the tube.

3.3 Place tube in a thermocycler and run the following program: lid 50℃; 37℃, 30 min, 4℃, hold.

4. RNA Purification

4.1 Equilibrate the Hieff NGS® RNA Cleaner(Cat#12602) to room temperature and resuspend the beads thoroughly by vortexing before use.

4.2 Add 110 µL (2.2×) beads to the RNA solution from Step 3.3 and mix thoroughly by pipetting up and down at least 10 times.

4.3 Incubate for 5 minutes on ice to bind RNA to the beads.

4.4 Place the tube on a magnetic rack to separate the beads from the supernatant. After the solution is clear(about 3 mins), discard the supernatant. Be careful not to disturb the beads which contain the RNA.

4.5 Add 200 µL of freshly prepared 80% ethanol to the tube while in the magnetic rack. Incubate at room temperature for 30 seconds and then discard the supernatant. Be careful not to disturb the beads which contain the RNA.

4.6 Repeat Step 4.5 once for a total of two washes.

4.7 Remove residual ethanol and air dry the beads for up to 5 minutes while the tube is on the magnetic rack with the lid open.

4.8 Remove the tube from the magnetic rack. Elute the RNA from the beads by adding 11 μL of Nuclease-free Water. Mix thoroughly by pipetting up and down at least 10 times and briefly spin the tube.

4.9 Incubate for 2 minutes at room temperature, Place the tube on the magnetic rack until the solution is clear (~ 3 minutes).

4.10 Remove 10 µL of the supernatant and transfer to a nuclease-free tube.

Note: If you need to stop at this point, samples can be stored at –80°C.

15221439726

15221439726