Biology Reference
In-Depth Information
2. Materials,
Instruments, and
Software
1. Rat blood and brain tissue.
2. Blood collection tubes containing EDTA (Cat. #: 367856,
Becton Dickinson and Company, Franklin Lakes, NJ).
3. RNA Later (Cat. #: AM7020, Ambion Inc., Austin, TX).
2.1. Sample (Blood
and Brain) Preparation
2.2. Total RNA
(Including miRNA)
Isolation
1. RNA Later preserved blood and brain tissue.
2. Mouse Ribopure-Blood RNA isolation kit (Cat. #: AM1951,
Ambion Inc.).
3. mir Vana miRNA isolation kit (Cat. #: AM1560, Ambion Inc.).
1. RNA samples.
2. NanoDrop-1000
2.3. Detection of
miRNA Concentration
and Integrity
spectrophotometer
(Thermo
Sci.,
Wilmington, DE).
3. Agilent-2100 Bioanalyzer (Agilent Technologies, Santa Clara,
CA).
4. Agilent RNA 6000 Nano kit (Cat. #: 50671511, Agilent
Technologies).
2.4. Stem-Loop
Reverse Transcription
Reaction
1. Good-quality RNA samples.
2. Megaplex RT Primers, Rodent Pool A (Cat. #: 4401090,
Applied Biosystems, Foster City, CA).
3. TaqMan miRNA RT kits (Cat. #: 4366596, Applied
Biosystems).
4. Thermocycler—GeneAmp PCR System 9700 (Applied
Biosystems).
1. Stem-loop RT products.
2. Megaplex PreAmp Primers, Rodent Pool A (Cat. #: 4399203,
Applied Biosystems).
3. TaqMan PreAmp Master Mix (Cat. #: 4391128, Applied
Biosystems).
4. Thermocycler—GeneAmp PCR System 9700 (Applied
Biosystems).
2.5. Preamplifi cation
Reaction
2.6. Real-Time PCR
Reaction
1. Stem-loop RT products, or diluted PA products.
2. TaqMan Universal PCR Master Mix (Cat. #: 4324018 FG,
Applied Biosystems).
3. TaqMan rodent miRNA fl uidic cards (Cat. #: 4398967,
Applied Biosystems).
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