MicroRNA Expression Profi ling of Rat Blood and Brain Tissues: TaqMan Real-Time PCR MicroRNA Assays - Animal Models of Acute Neurological Injuries: Injury and Mechanistic Assessments - page 158

10 min at 95°C, 2 min at 55°C, 2 min at 72°C, followed by 12
cycles of 95°C for 15 s and 60°C for 4 min, and then held at
4°C.
4. All RT reactions, including no-template controls and RT minus
controls, are run in duplicate.

1. If the starting total RNA of the sample is in the range of
1-350 ng, a preamplifi cation (PA) reaction is required prior to
performing the real-time PCR reaction.
2. Each PA reaction contains:
(a) 2.5 ml stem-loop RT product.
(b) 22.5 ml PA reaction mix.
2.5

3.5. Preamplifi cation
Reaction

ml Megaplex PA Primers, Rodent Pool A (2×).
12.5

●

ml TaqMan PreAmp Master Mix (2×).

●

● ml nuclease-free water.
3. The 25 ml reactions are incubated in an Applied Biosystems
9700 Thermocycler in either a 96-well plate or 8-tube strips
for 10 min at 95°C, 2 min at 55°C, 2 min at 72°C, followed by
12 cycles of 95°C for 15 s and 60°C for 4 min, and then held
at 4°C.
4. Dilute the PA products.
(a) Remove the 96-well plate or 8-tube strips from the
thermocycler.
(b) Add 75 ml of TE buffer (0.1×), pH 8.0, to each well or
tube.
(c) Seal the plate or tubes, then invert six times to mix, and
spin down briefl y.

7.5

3.6. Real-Time PCR
Reaction

1. TaqMan 384-well rodent miRNA fl uidic cards are used to pro-
fi le 381 mature miRNAs, with specifi c primers and probes pre-
plated in each well.
2. U6 is used as the endogenous control in quadruplicate and
one assay for a target not found in the rat is included as a nega-
tive control.
3. Each real-time PCR reaction contains:
(a) 6 ml stem-loop RT or 9 ml diluted PA product.
(b) 450 ml TaqMan Universal PCR Master Mix.
(c) Nuclease-free water to bring the reaction to 900 ml.
4. Combine the above components into one tube and mix well.
5. Dispense 100 ml of the PCR reaction mix into each port of the
TaqMan miRNA fl uidic card (Fig. 1 ).