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 156

4. Sorvall Legend T Centrifuge (Fisher Scientifi c, Pittsburgh, PA)
equipped with Heraeus swinging buckets for Heraeus card
adapters (Cat. #: 75015686, Fisher Scientifi c).
5. Applied Biosystems 7900HT real-time PCR System equipped
with a heating block for the fl uidic card (Applied Biosystems).
6. Applied Biosystems SDS 2.3 software (Cat. #: 4361171,
Applied Biosystems).

2.7. Statistical
Analysis

1. RQ Manager 1.2 (supplied with Applied Biosystems SDS 2.3
software, Cat. #: 4361171, Applied Biosystems).
2. Partek Genomics Suite (Partek, St. Louis, MI).
3. Genespring GX software (Agilent Technologies).

1. miRNA function prediction software: MetA Mir:target
Inference (MAMI), ( http://mami.med.harvard.edu ).
2. Ingenuity pathway analysis (IPA) software (Ingenuity Systems,
Redwood City, CA).

2.8. Analysis of
Biological Function
and Signifi cance

3. TaqMan miRNA
Array Procedures

3.1. Blood and Brain
Sample Preparation

1. Rat blood (0.5 ml) is drawn from the tail vein into EDTA
tubes and immediately mixed with 1.3 ml RNA Later and then
stored at −80°C. Note that alternative products designed to
stabilize RNA in blood exist and may also be used.
2. Under anesthesia, rat brains are removed and dissected as rap-
idly as possible and immersed into the RNA Later for an over-
night period at 4°C. On the following day, the brain tissue is
removed from the RNA Later and stored in a −80-C freezer.

1. Total RNA (including miRNA) is isolated from blood using
the Mouse Ribopure-Blood RNA isolation kit ( 3 ).
2. Total RNA (including miRNA) is isolated from the selected
brain region using the mir Vana miRNA isolation kit or the
miRNeasy Mini Kit (Qiagen, Valencia, CA) ( 3 ).
3. These methods of RNA isolation are described in the manufac-
turer's protocol.

3.2. Isolation of Total
RNA (Including
miRNA)

3.3. Detection of
miRNA Concentration
and Integrity

1. The concentration of total RNA was determined using the
NanoDrop-1000 spectrophotometer. This quick and reliable
method uses a minimal amount of the RNA sample, preserving
most of the sample for other analyses.
2. The integrity of total RNA was determined by running samples
on Agilent RNA 6000 Nano chips using the Agilent-2100