Fresh striatal, cortical, and cerebellar tissue was dissected from mice at 2 months of age, snap-frozen in liquid nitrogen and stored at −80° C. Tissue was homogenized in Qiazol buffer (Qiagen) before total RNA isolation using the QiaGen RNeasy mini kit (Qiagen) and RNA was quantified using a Nanodrop 1000. RNA was reverse transcribed using MMLV-RT (Invitrogen) and oligo-dT primers (Invitrogen) and cDNA was stored at −20°C. qPCR was performed using SsoFast Probes Supermix (Bio-Rad) with a corresponding cycler program using a CFX 96 qPCR system (Bio-Rad). Primer-probe set gene expression assays for the Htt intron 1 sequences were 347F-431R-371P and for spliced Htt were −19F-ex2R-ex2P as detailed in Table 2 . Housekeeping reference genes were Atp5b, Eif4a2 and Ubc (striatum and cerebellum) and Atp5b, Eif4a2, Canx and Rpl13a (cortex) from Primer Design (Benn et al., 2008 (link)). The level of intronic sequences was determined by normalizing to housekeeping genes. The relative levels of the spliced transcript was determined by the 2−ΔΔCt method (Benn et al., 2008 (link)).
Protocol detail
Quantitative Analysis of Huntingtin Expression
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Qiazol buffer
QiaGen RNeasy mini kit
Nanodrop 1000
MMLV-RT
oligo-dT primers
SsoFast Probes Supermix
CFX 96 qPCR system
Assays
Atp5b
Buffer
Cdna
Cerebellum
Cortex
Cortical
Frozen
Gene expression
Housekeeping genes
Intronic
Isolation
Mice
Nitrogen
Oligo dt
Primer
Striatum
Tissue
Corresponding Organization : UK Dementia Research Institute
Other organizations : University of Tartu, University of Virginia, CHDI Foundation
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Variable analysis
independent variables
- Age of mice at tissue collection (2 months)
- Expression levels of Htt intron 1 sequences
- Relative levels of spliced Htt transcript
- Tissue types (striatal, cortical, and cerebellar)
- Tissue storage conditions (snap-frozen in liquid nitrogen and stored at −80° C)
- RNA isolation method (QiaGen RNeasy mini kit)
- RNA quantification method (Nanodrop 1000)
- Reverse transcription method (MMLV-RT and oligo-dT primers)
- QPCR method (SsoFast Probes Supermix, CFX 96 qPCR system)
- Primer-probe sets for Htt intron 1 and spliced Htt
- Housekeeping reference genes (Atp5b, Eif4a2, Ubc for striatum and cerebellum; Atp5b, Eif4a2, Canx, Rpl13a for cortex)
- Not explicitly mentioned
- Not explicitly mentioned
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