TruSeq Targeted RNA Expression Kit FAQs

TruSeq Targeted RNA Expression is a library prep kit that allows efficient, multiplexed gene expression profiling for 12–1000 targets per sample and up to 384 samples in one MiSeq System run.

Select the RNA Resequencing category, and then select the Targeted RNA application.

This tube contains the PCR primers used to amplify the cDNA amplicons. The primer sequences include sequencing primer binding sites and the indices/barcodes.

On average, the gap size is 15 bases, but can be as small as 1 and as large as 43 bases. The average insert size is 67 bp. The average length of the ULSO and DLSO are 25 bases, not including the PCR primer binding sites.

Use the Export Manfest function in Project Dashboard of your DesignStudio project to download manifest files. The manifest file is available after your order ships, not when the order is placed or designed in DesignStudio.

TruSeq Targeted RNA Expression kits currently support only human, mouse, and rat organisms.

Analyzing TruSeq Targeted RNA Expression data requires a manifest file (*.txt). The manifest file contains the probe and target sequences for your order. MiSeq Reporter uses it for on-instrument alignment.

For some library preparations, AMPure XP beads are user-supplied from Beckman-Coulter Genomics. See the appropriate TruSeq DNA or RNA library prep guide for more information.

Human dbSNP 135 was used for the designs and the following criteria were considered for cSNP assays:

• cSNP must be common (ie, those with ≥ 1% MAF have been included)
• There should be no other cSNP in the probe design area
• Only 1 SNP per target is allowable
• cSNP must be covered in all transcripts to a gene

Use DesignStudio to order probes. You will need a MyIllumina account.

An add-on project uses a fixed content pool, which is either a fixed or custom panel, that you can add 12–1000 custom targets to.

Assays marked validated in DesignStudio have been shown to give results consistent with fold changes observed by RNA-Seq in testing across human tissues.

No, DesignStudio assays target splice junction sites across the entire transcript. Additional designs are available for cSNPs or gene fusions. The following databases were used for assay designs:

Your order can contain 12–1000 probes.

The TruSeq Targeted RNA Expression assay consists of predesigned probes. Once a gene is selected, DesignStudio provides a list of predesigned probes. Due to the predesigned nature of these probes, DesignStudio design time is negligible. Options for probes include:

• Transcript specific
• 5' end
• 3' end
• cSNP

Illumina internal testing found that > 90% of nonvalidated assays correlate with RNA-Seq data. Assays marked validated have demonstrated results consistent with fold changes observed by RNA-Seq in testing across human tissues.

Every effort was made to include as many transcripts as possible. For human and mouse transcripts, refseq transcript models were used together with the hg19 and mm9 +mm10 genomes, respectively.

Fixed content projects are presynthesized oligo pools that you can order. The pools have been wet-bench tested and designed against various cellular and disease-causing pathways.

Download the TruSeq Targeted RNA Expression Gene Fusion List for a list of the gene fusions included in TruSeq Targeted RNA Expression.

Illumina has tested up to 500 ng of high-quality RNA in starting material. However, no improvement in yield is seen in the final amount of RNA generated beyond 200 ng of starting material for high-quality RNA.

Samples are quantified after pooling. 

Yes, Illumina recommends using 100–200 ng degraded or FFPE samples with an average fragment size of ≥ 200 bp for optimal hybridization. Check the total RNA integrity following isolation on an Agilent Technologies 2100 Bioanalyzer using an RNA 6000 Nano Chip. For more information, see the RNA Input Recommendations section of the TruSeq Targeted RNA Expression Reference Guide.

In order to normalize your data, identify a gene from RNA-Seq or Microarray data that is constant across the samples being tested. Include assays that target this gene in your TOP panel when using DesignStudio. Additionally, this gene needs to be identified in the sample sheet, in a column with the header “Normalize”, so MiSeq Reporter will use it to normalize the data.

Illumina recommends preparation of 16–96 samples at a time. Smaller batch sizes are supported, as long as do not undergo more than five freeze-thaw cycles. Larger batch sizes may be performed by more experienced users, taking care that the OB1 beads do not dry during purification steps.

Resuspension Buffer should be stored at -25ºC to -15ºC when first received. After the initial thaw, the reagent can be stored at 2ºC to 8ºC for use throughout the protocol.

This is dependent on the experiment plexity and MiSeq version. Refer to the TruSeq Targeted RNA Expression technical note for information on how to design an experiment and determine the appropriate number of samples to be run on a flow cell.

TruSeq Targeted RNA Expression is currently supported on the MiSeq System. The on-instrument analysis workflow in MiSeq Reporter supports this protocol.

Set up a 1 x 50, dual-indexed run to sequence TruSeq Targeted RNA Expression libraries.

Yes, however Targeted RNA Expression libraries require 50 cycles of sequencing (single read), plus dual indexes.

Perform a region analysis on the Bioanalyzer and define the region from 150–250 bases to get molarity for the entire sample.

This information can be found in the .bam files. However, MiSeq Reporter Software does not perform variant calling or generate .vcf files. cSNPs can be analyzed and visualized using the Illumina Genome Viewer in GenomeStudio.

Yes, if the data from different runs is normalized with same normalizer genes. The raw data from different runs cannot be merged together into a single dataset. For more information, see the MiSeq Reporter Software Guide.

Use MiSeq Reporter for on-instrument analysis. This software provides analysis of replicates and allows for pair-wise comparison of targeted regions.