Abi prism 7000 sequence detection

RNA from cultured cells was isolated using RNeasy® Mini Kit (Qiagen), followed by cDNA synthesis using GoScript^TM Reverse Transcription System (Promega). Quantitative reverse transcriptase PCR was performed with GoTaq® qPCR Master Mix (Promega) on the ABI prism 7000 sequence detection system (Applied Biosystems, Eugene, OR). The results for each sample were normalized to the 18SrRNA. All assays were conducted at least three times.

Saliva samples were collected from each participant using the Oragene DNA Collection Kit (DNA Genotek, Ottawa, ON, Canada) and sent to the Translational Genomics Research Institute in Phoenix, Arizona for genotyping of KIBRA rs17070145, APOE rs429358, and APOE rs7412. Genotyping was carried out using TaqMan allelic discrimination (Applied Biosystems, Foster City, CA, United States) and ABI Prism 7000 sequence detection (Applied Biosystems, Foster City, CA, United States) as described in Corneveaux et al. (2010) (link). KIBRA rs17070145 (GenBank accession number NC_000005 GPC_000001297, version number NC_000005.10) genotypes were generated using the KASP chemistry (LGC, Middlesex, United Kingdom). In brief, KASP reactions are comprised of sample DNA, KASP Master Mix, and KASP Assay Mix which contains variant specific probe combinations. The DNA samples are then subjected to polymerase chain reaction to generate fluorescent signals that indicate genotypes for the variants of interest. Amplifications errors resulted in undetermined genotypes for four individuals who were not included in the present study. Genotype distributions for our sample were 0.14, 0.37, and 0.48 for TT homozygotes, TC heterozygotes, and CC homozygotes, respectively. Our sample did not violate Hardy–Weinberg equilibrium [χ²(1) = 3.03, p = 0.08].

The isolation of total RNA was conducted using TRIzol reagent (Invitrogen). U6 snRNA or β‐actin mRNA was chosen as internal reference to normalize the expression of miR‐335‐5p or BCL2L2 mRNA, respectively. To examine the expression of miR‐335‐5p, stem‐loop‐specific primer was utilized for amplification, while DNA Reverse Transcription Kit (Applied Biosystems, Foster City, CA, USA) was used to perform the reverse transcription so as to determine the expression of BCL2L2 mRNA. Quantitative real‐time PCR was performed using SYBR Select Master Mix in ABI Prism 7000 Sequence Detection (Applied Biosystems). Fold changes were calculated by 2^−ΔΔCt. Primers used were manifested in Table 1.