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Genomic and RNA Profiling Core Facility

Houston, Texas

BCM faculty, staff and trainees are the heart of the organization.
Genomic and RNA Profiling Core
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mRNA and Total RNA Sequencing Services

mRNA and total RNA sequencing offers gene expression profiling using RNA. Beyond gene expression changes, RNA sequencing can provide information about alternative transcripts, alternative splicing, gene fusions, allele-specific expression and novel transcripts.

Prior to constructing an Illumina sequencing library, total RNA quantity and quality is validated by running an aliquot of the sample on the NanoDrop spectrophotometer and an Agilent Bioanalyzer RNA chip. The user will be given a copy of these files following sample quality check and prior to library preparation. DNAse treatment of RNA is required to avoid false-positive results in downstream applications. Failed samples must be signed off by the PI.

Illumina TruSeq RNA Sample Preparation Kit v2

TruSeq RNA sample prep kit converts the poly-A containing mRNA in total RNA into a cDNA library using poly-T oligo-attached magnetic bead selection. Following mRNA purification, the RNA is chemically fragmented prior to reverse transcription and cDNA generation. The fragmentation step results in an RNA-seq library that includes inserts that range in size from approximately 100-400 bp. The average insert size in an Illumina TruSeq RNA sequencing library is approximately 200 bp. The cDNA fragments then go through an end repair process, the addition of a single ‘A’ base to the 3’ end and then ligation of the adapters. Finally, the products are purified and enriched with PCR to create the final double stranded cDNA library. Up to 24 samples can be multiplexed together in a single flow cell lane.

Illumina TruSeq Stranded mRNA Sample Preparation Kit

TruSeq stranded mRNA identifies from which of the two DNA strands an RNA transcript was derived and offers strand orientation for detection of antisense transcription, providing information about regulatory relationships. TruSeq stranded RNA sample prep kit converts the poly-A containing mRNA in total RNA into a cDNA library using poly-T oligo-attached magnetic bead selection. Following mRNA purification, the RNA is chemically fragmented prior to reverse transcription and cDNA generation. The fragmentation step results in an RNA-seq library that includes inserts that range in size from approximately 100-400 bp. The average insert size in an Illumina TruSeq RNA sequencing library is approximately 200 bp. During second strand synthesis, dUTP is incorporated in place of dTTP, which cannot be amplified during enrichment. The cDNA fragments then go through an end repair process, the addition of a single ‘A’ base to the 3’ end and then ligation of the adapters. Finally, the products are purified and enriched with PCR to create the final double stranded cDNA library. Up to 24 samples can be multiplexed together in a single flow cell lane.


Illumina TruSeq Total RNA Sample Preparation Kit

Whole transcriptome analysis using ribosomal reduction provides detection of both coding and non-coding RNA, along with other long intergenic noncoding RNA (lincRNA), small nuclear RNA (snRNA), and small nucleolar RNA (snoRNA). Whereas mRNA sequencing requires high quality total RNA, the TruSeq total RNA can provide stranded information using degraded or formalin-fixed paraffin embedded (FFPE) samples and is optimized for human, mouse, and rat samples, but can be customized for most sample types. Ribo-Zero ribosomal reduction prior to library construction removes cytoplasmic ribosomal RNA (rRNA) and mitochondrial rRNA by hybridizing the rRNA to biotinylated capture probes. Following ribosomal reduction and cleanup, the RNA is chemically fragmented prior to reverse transcription and cDNA generation. The fragmentation step results in an RNA-seq library that includes inserts that range in size from approximately 100-400 bp. The average insert size in an Illumina TruSeq RNA sequencing library is approximately 200 bp. The cDNA fragments then go through an end repair process, the addition of a single ‘A’ base to the 3’ end and then ligation of the adapters. Finally, the products are purified and enriched with PCR to create the final double stranded cDNA library. Up to 24 samples can be multiplexed together in a single flow cell lane.

Illumina RNA-Seq Input Amounts
Illumina Kit GARP Recommended Minimum total RNA input GARP Recommended Minimum Concentration RNA Quality
mRNA Seq
1-2 μg (0.1 μg)*
84 ng/µL (34 ng/µL)*
High Quality
Total RNA Seq
500 ng (100 ng)*
34 ng/µL (17 ng/µL)* High Quality, degraded or FFPE
*Minimum Assay Requirements

* DNase treatment of RNA is required to avoid false positives in downstream applications.*


Epicentre RNA Sequencing Ribo-Zero Magnetic Kit

Whole transcriptome analysis using ribosomal reduction provides detection of both coding and non-coding RNA, along with other long intergenic noncoding RNA (lincRNA), small nuclear RNA (snRNA), and small nucleolar RNA (snoRNA). The Epicentre Ribo-Zero magnetic kit removes cytoplasmic RNA from total RNA using a hybridization/bead capture procedure that selectively binds rRNA on biotinylated capture probes, leaving behind the rRNA-depleted RNA in solution. The gold kit provides additional removal of cytoplasmic ribosomal RNA. These kits are pre-made for human/mouse/rat, bacteria, plant or mammalian blood, but can be customized for other species.

Epicentre ScriptSeq v2 RNA-Seq Library Preparation Kit

The ScriptSeq v2 kit prepares RNA libraries from rRNA-depleted or poly-A enriched RNA from any animal, plant or bacterial species. The data produced will be directional mRNA, or mRNA plus ncRNA, depending on RNA input type. An additional kit must be purchased prior to library prep in conjunction with this kit. Up to 48 samples can be multiplexed together in a single flow cell lane.

Epicenter RNA-Seq Input Amounts
Epicenter Kit GARP Recommended Minimum total RNA Input GARP Recommended Minimum Concentration RNA Quality
Ribo-Zero
4 μg (1 μg)*
155 ng/μl (40 ng/μl)*
High Quality, degraded or FFPE
ScriptSeq v2
25 ng (500 pg)*
2.8 ng/μl (0.56 ng/μl)* rRNA-deleted or poly-A selected RNA
*Minimum Assay Requirements

* DNase treatment of RNA is required to avoid false positives in downstream applications.*


Other RNA-Seq Sample Prep Kits

NuGEN Ovation RNA-Seq v2

NuGEN Ovation RNA-Seq v2 converts total RNA to cDNA using a polyA and random priming method, which is then amplified using SPIA amplification. This procedure will amplify both mRNA and non-polyadenylated transcripts that will be sheared and used for library preparation. This kit must be used in conjunction with a library preparation kit.

NuGEN Ovation RNA-Seq FFPE System

NuGEN Ovation FFPE converts FFPE-derived total RNA to cDNA, which is then amplified using a polyA and random priming method, which is then amplified using SPIA amplification, making it ideal for severely degraded or chemically modified RNA. This procedure will amplify both mRNA and non-polyadenylated transcripts that will be used for library preparation. This kit must be used in conjunction with a library preparation kit.

NuGEN Ovation Prokaryotic RNA-Seq System

NuGEN Ovation Prokaryotic RNA-Seq allows for whole transcriptome profiling of microbial species and microbiome samples. Total RNA is first converted to double stranded cDNA using selective priming, which can then be used as input with NuGEN’s Ovation Ultralow Library System.

NuGEN RNA-Seq Input Amounts
Epicenter Kit GARP Recommended Minimum total RNA input GARP Recommended Minimum Concentration RNA Quality
Ovation RNA-Seq v2
10 ng (500 pg)*
2 ng/μl (0.05 ng/μl)*
High quality, degraded or FFPE
Ovation RNA-Seq FFPE 150 ng (100 ng)* 30 ng/μl (20 ng/μl)* Severely degraded or chemically modified Total RNA
Ovation Prokaryotic RNA-Seq
500 ng
62.5 ng/μl Severely degraded or chemically modified Total RNA
*Minimum Assay Requirements

* DNase treatment of RNA is required to avoid false positives in downstream applications.*


Agilent SureSelect Strand-Specific mRNA Library preparation
Agilent’s SureSelect Strand Specific RNA Library Preparation Kit offers a highly sensitive, strand-specific method for preparing libraries for mRNA for targeted RNA sequencing. The RNA library prep kit provides high strand specificity with greater uniformity of coverage across the entire transcript, enabling a greater understanding of gene regulation. It also provides unmatched accuracy for better discovery of novel transcripts and gene fusions. The kit has been designed to maintain high library complexity even with small amounts of starting material, as low as 50ng of total RNA, for studies with limited samples amounts.
Agilent SureSelect Strand-Specific mRNA Input Amounts
Total RNA Input
Minimum Concentration
50 ng - 4 μg
2 ng/μl - 160 ng/μl

Agilent SureSelect RNA Target Enrichment
Agilent combines their SureSelect Strand-Specific mRNA protocol with their SureSelect Target Enrichment system with SureSelect RNA Target Enrichment kits. Custom design of bait panels allow researchers to sequence targeted RNA enabling them to focus on specific transcripts of interest. Sequencing smaller targeted regions further reduces cost by allowing multiplexing of more samples without compromising performance. With Agilent’s SureSelect RNA Target Enrichment researchers can precisely measure expression levels of specific genes and their isoforms.
Agilent SureSelectXT RNA Target Enrichment Input Amounts
Total RNA Input
Minimum Concentration
200 ng - 4 μg
8 ng/μl - 160 ng/μl

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