Reverse Transcribe cDNA

In this step, the ligated miRNAs are reverse transcribed to cDNA using a reverse transcription primer with a unique molecular identifier (UMI).

The RT primer binds to a region of the 3' adapter and facilitates conversion of the 3'/5' ligated miRNAs into cDNA. The primer also assigns a UMI to every miRNA molecule. During reverse transcription, a universal sequence is added.

If you are working with low total RNA input amounts or with serum or plasma samples, you must dilute the miRNA RT Primer 2. Instructions are provided in the following Preparation section.

• MRNH (miRNA RNase Inhibitor)
• MRTE (miRNA RT Enzyme)
• RTB (miRNA RT Buffer)
• RTP2 (miRNA RT Primer 2)
• URTI (UDI RT Initiator)
• [Low input, serum, or plasma RNA] NFW (Nuclease-free water)
• Microcentrifuge tubes, 1.5–2 ml
• MRNH and MRTE
– Remove from the -25°C to -15ºC freezer immediately before preparing the Master Mix. Place on ice.
– Aliquot into smaller volumes upon first thaw to avoid repeated freeze-thaw cycles.
– Avoid vortexing.
– Return to the freezer immediately after use.
1. Prepare the following consumables:

Reagent

Storage

Instructions

RTB

-25°C to -15°C

Thaw at room temperature.

RTP2

-25°C to -15°C

Thaw at room temperature.

URTI

-25°C to -15°C

Thaw at room temperature.

2. Flick each tube to mix, and then centrifuge briefly to collect residual liquid from the sides of the tube. Keep at room temperature.
3. If you are working with low RNA inputs or with serum or plasma samples, dilute the RTP2 as follows.

Add nuclease-free water to the RTP2, as specified in the following table.

Template RNA Input (Total RNA)

Adapter Dilution

500 ng

Use undiluted

100 ng

Use undiluted

10 ng

Dilute 1:5

1 ng

Dilute 1:20

Serum or plasma

Dilute 1:5
4. Save the following RT1 program on the thermal cycler:
• Choose the preheated lid option and set to 100°C
• Reaction volume is 42 µl
• 75°C for 2 minutes
• 70°C for 2 minutes
• 65°C for 2 minutes
• 60°C for 2 minutes
• 55°C for 2 minutes
• 37°C for 5 minutes
• 25°C for 5 minutes
• Hold at 4°C
5. Save the following RT2 program on the thermal cycler:
• Choose the preheated lid option and set to 100°C
• Reaction volume is 60 µl
• Incubate at 50°C for 1 hour
• Incubate at 70ºC for 15 minutes
• 4°C for 5 minutes
• Hold at 4°C
6. Immediately before preparing the reverse transcription reaction, prepare the following consumables:

Reagent

Storage

Instructions

MRNH

-25°C to -15°C

Place on ice. Return to the freezer immediately after use.

MRTE

-25°C to -15°C

Place on ice. Return to the freezer immediately after use.
1. Add 2 µl URTI to each tube containing the 5' ligation reaction.
2. Centrifuge briefly.
3. Pipette up and down 15–20 times to mix, and then centrifuge briefly a second time.
4. Place on the thermal cycler and run the RT1 program. Hold at 4°C until ready to prepare the reverse transcription reaction.
5. Working on ice, prepare the reverse transcription reaction as follows.

Add the following volumes to the tube containing the 5' ligation reaction in the order listed. For multiple reactions, multiply each volume by the number of samples plus 10% extra.

Component

Volume per Reaction (µl)

5' ligation reaction + URTI

(already in tube)

42

RTP2 (dilute as needed)*

2

Nuclease-free water

2

RTB

12

MRNH

1

MRTE

1

Total volume per reaction

60

* For low input, serum, and plasma RNA, the RTP2 must be diluted (refer to Preparation section).

Avoid vortexing the reverse transcription reactions.

6. Place on the thermal cycler and run the RT2 program.
7. Proceed immediately to Prepare Illumina miRNA Beads. You can perform this step while the reverse transcription reactions are incubating.