Biology Reference
In-Depth Information
Whenever possible, use sterile, disposable plastic ware, because
these materials are generally RNase-free.
●
Glassware should be baked at 180-200 °C for at least 4 h.
●
Solutions (water and other solutions) should be treated by add-
ing diethyl pyrocarbonate (DEPC) to 0.1 vol %. Following incu-
bation overnight at room temperature, the treated solutions
should be autoclaved for 30 min to remove any trace of DEPC.
●
PCR assays require special laboratory practices to avoid false-
positive amplifi cations because a single DNA molecule can be
detected by this technique. When preparing samples for PCR
amplifi cation, the following precautions should be taken:
Prevent aerosols.
●
Wear clean gloves (not previously worn while handling ampli-
fi ed material or used during sample preparation).
●
Designate a PCR setup area and never bring amplifi ed PCR
product into this area.
●
Open and close all sample tubes carefully, try not to splash
PCR samples.
●
Keep reactions and components capped as much as possible.
●
Use ultrapure water (18 M
Ω
cm at 25 °C).
●
Store all solutions in designated areas free of amplifi ed DNA or
artifi cial templates.
●
1. Collect desired tissues and snap freeze in liquid nitrogen and
store at −80 °C.
2.1 Tissue
2.2 RNA Extraction
1. RNAqueous
®
-Micro, Micro Scale RNA Isolation Kit (Ambion).
2.3 cDNA Synthesis
1. TaqMan
®
MicroRNA Assays (ABI) for specifi c miRNAs.
2. TaqMan
®
MicroRNA Reverse Transcription Kit.
3. miScript Reverse Transcription Kit (Qiagen).
2.4 qRT-PCR
1. TaqMan
®
MicroRNA Assays (ABI) for specifi c miRNAs
(
see
Subheading
2.3
).
2. TaqMan
®
Universal PCR Master Mix 2×, No AmpErase
®
UNG (ABI).
3. miScript SYBR Green PCR Kit (Qiagen).
4. miScript Precursor Assays (Qiagen).
2.5 Equipment
1. Real-time thermal cycler compatible with the selected
technology.
2. Spectrophotometer for low sample volumes (1
L).
3. Agilent 2100 Bioanalyzer (Agilent Technologies).
μ
