Institute for Immunity, Transplantation and Infection

The Human Immune Monitoring Center (HIMC)

For more information

Contact:

Xuhuai Ji, Genomics Manager
Email » [xuhuai.ji]

Address:
Human Immune Monitoring Center
CCSR Building, Room 0128
269 Campus Drive
Stanford, CA 94305

phone:  650-723-5050
fax:  650-498-6345

Genomics

The HIMC offers RNA and DNA extraction, Bioanalyzer services, gene expression and miRNA microarrays, and microfluidic qPCR arrays for human and mouse.

RNA and DNA extraction and quality check:  Nucleic acid extraction can be done from PAXgene RNA or DNA tubes, or from samples resuspended in Trizol.  Quality/integrity is checked using an Agilent Bioanalyzer chip (small, nano, or pico scale), which automatically determines an RNA Integrity Number (RIN) as well as a visual depiction of electrophoretic results.  The Bioanalyzer can also be used to determine total protein concentration of cell lysates of other protein samples.

Microarrays:  We offer a custom "HIMChip" microarray containing ~7000 unique probles for over 3000 human immune-related genes (click here for the list).  Genes were derived from the Immunology Database and Analysis Portal, based on the Entrez Gene, UniProt, KEGG, and various literature sources.  This is a custom Agilent SurePrint HD 8x15K format array, with 8 arrays available per slide.  We also offer whole-genome microarrays, including human and mouse miRNA arrays, on the Agilent or Illumina platform, in collaboration with the Stanford Functional Genomics Facility (SFGF).

Microfluidic qPCR arrays:  The Fluidigm BioMark system allows for the rapid construction of qPCR arrays on a microfluidic chip, using minimal samples volumes and minimal pipetting.  The system is compatible with single-cell qPCR.  A set of primers for B- and T-cell specific genes is available in the HIMC, and others can be purchased by customers for use on this platform.

User Instructions

1.  Planning your assay:  Please keep in mind the architecture of microarray chips (8 or 12 arrays per slide) when planning experiments and batches.  Please consult with us regarding optimal experimental design (1- or 2-color arrays, replicates, controls).

2.  Collecting your samples:  Good RNA quality and yield are dependent on proper sample handling.  Please see the protocols at the bottom of this page, or consult with us before delivering samples.  Avoiding sample contamination is also critical for qPCR experiments.

3.  Running your samples:  An online order is required before samples can be run. Turnaround time varies depending on the size of your project and the current workload; smaller projects can usually be completed within 3-4 weeks of order placement and sample delivery.

4.  Data analysis:  You will be notified by email when assays are completed.  Summary reports are provided for Bioanalyzer runs and microrray results.  Microarray raw data files are available for download via Dropbox, or by file transfer in the HIMC (CCSR room 0128).  Additional data analysis services are available for a fee (see our price list for details).

Protocols

1.  Short protocol descriptions, suitable for publications or grant applications:

RNA extraction and microarray--whole blood (PAXgene), Illumina microarray:  RNA sampling and extraction: Blood was collected directly into PAXgene Blood RNA Tube (BD PreAnalytix) using a 21-gauge butterfly needle and catheter so that tubes could be held low and vertical to maintain the vacuum on top of the stabilization solution. Tubes were inverted 10 times. Each tube drew a maximum of 2.5 ml of blood. Samples were incubated in collection tubes at room temperature then stored at -80C within 4h. Total RNA was isolated according to the manufacturer’s Instructions by using a PAXgene RNA blood kit (Qiagen).The entire procedure was carried out at room temperature with the QIAcube automated robot (Qiagen) . Total RNA yield was assessed using the Thermo Scientific NanoDrop 1000
micro-volume spectrophotometer (absorbance at 260 nm and the ratio of 260/280 and 260/230). RNA integrity was assessed using the Agilent’s Bioanalyzer NANO Lab-on-Chip instrument (Agilent).

Microarray processing and analysis: Biotinylated, amplified antisense complementary RNA (cRNA) targets were prepared from 200 to 250 ng of the total RNA using the Illumina RNA amplification kit (Applied Biosystems/Ambion). Seven hundred and fifty nanograms of labeled cRNA was hybridized overnight to Illumina Human HT-12 V3 BeadChip arrays (Illumina), which contained >48,000 probes. The arrays were then washed, blocked, stained and scanned on an Illumina BeadStation 500 following the manufacturer’s protocols. BeadStudio/GenomeStudio software (Illumina) was used to generate signal intensity values from the scans. For normalization, the software was used to subtract background and scale average signal intensity for each sample to the global average signal intensity for all samples. A gene expression analysis software program, GeneSpring GX version 7.3.1 (Agilent Technologies), was used to perform further normalization. Transcripts meeting the filtering criteria were subjected to hierarchical clustering using GeneSpring.

RNA extraction and microarray--PBMC or sorted cells, Agilent microarray:  RNA sampling and extraction: PBMC or sorted cell populations (< 1x10^7 viable cells) were collected in 1ml TRIzol (Invitrogen) and stored at -80c until use). Total RNA was isolated according to the TRIzol protocol (Invitrogen) or RNeasy Mini Kit (Qiagen). For using the RNeasy Mini Kit, the entire procedure was carried out at room temperature with the QIAcube automated robot (Qiagen) . Total RNA yield was assessed using the Thermo Scientific NanoDrop 1000 micro-volume spectrophotometer (absorbance at 260 nm and the ratio of 260/280 and 260/230). RNA integrity was assessed using the Agilent’s Bioanalyzer NANO Lab-on-Chip instrument (Agilent).

Microarray processing and analysis: Cy3 and/or Cy5 labeled, amplified antisense complementary RNA (cRNA) targets were prepared from 20 to 500 ng of the total RNA using the QuickAmp Labeling kit or the Low Input Quick Amp Labeling Kit (Agilent). 850 ug of labeled cRNA was hybridized overnight to Agilent Whole Human Genome 4 x 44 K slides, which contain 44,000 probes, including 19,596 Entrez Gene RNAs; or to SurePrint G3 Human Gene Expression 8x60k slides, which contain 60,000 probes, including 27,958 Entrez Gene RNAs and 7,419 lincRNAs. The arrays were then washed, blocked, stained and scanned on the Agilent microarray scanner following the manufacturer’s protocols. Data were extracted using Agilent Feature Extraction Software. Microarray normalization was performed by GeneSpring GX 11.0 software. Further statistical and bioinformatic analyses were done with Ingenuity Pathway Analysis software (Ingenuity Systems).Transcripts meeting the filtering criteria were subjected to hierarchical clustering using GeneSpring.

Biomark Microfluidic qPCR Array:  For 10-50 ng total RNA, reverse transcription of the RNA to cDNA was performed at 50°C for 15 minutes using the High Capacity Reverse Transcription kit (ABI). For cell samples, RT was performed directly on a 96-well PCR plate (ABI) containing lysis buffer (Invitrogen) by using SuperScript III One-Step RT-PCR System with PlatinumTaq (CellDirect kit, Invitrogen). PreAmp was performed on a thermocycler using the TaqMan PreAmp Master Mix Kit (Invitrogen) added to cDNA and pooled Taqman assays. RT enzyme was inactivated and the Taq polymerase reaction was started by bringing the sample to 95 °C for 2 minutes. The cDNA was preamplified by denaturing for 10 (total RNA) to 18 (single cell) cycles at 95°C for 15 seconds, annealing at 60°C for 4 minutes. The resulting cDNA product was diluted 1:2 with 1x TE buffer (Invitrogen). 2X Applied Biosystems Taqman Master Mix , Fluidigm Sample Loading Reagent, and preamplified cDNA were mixed and loaded into the 48.48 Dynamic Array (Fluidigm) sample inlets, followed by loading 10X assays into the assay inlets. Manufacturer’s instructions for chip priming, pipetting, mixing, and loading onto the BioMark system were followed. Real-time PCR was carried out with the following conditions: 10 min at 95°C, followed by 50 cycles of 15 sec at 95°C and 1 min at 60°C. Data was analyzed using Fluidigm software. All reactions were performed in duplicate or triplicate, and Ct values were normalized to the GAPDH or 18S positive control.

2. Detailed protocols and addtional information:

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