title: Sample 2_ME
organism: Homo sapiens
description: Protocols: The work described in this report is a study using targeted sequencing-based RNA expression analysis on formalin-fixed, paraffin-embedded (FFPE) tissue sections of human prostate. The radical prostatectomy specimens from the 15 male patients included in this study were labelled, weighed and formalin fixed. This included 15 samples from each of three different regions of interest - normal prostate tissue, cancerous tissue, and the microenvironment. An excision of 10mm2 was taken for normal prostate and cancerous regions, and an excision of 5mm2 was taken for microenvironment due to the smaller area available for this region across slides. The radical prostatectomy specimens from the 15 male patients included in this study were labelled, weighed and formalin fixed. Subsequently, the radical prostatectomy specimens were sliced in patient-specific moulds to assist in correlating and adjusting magnetic resonance images with histology. Before an excision of 10mm2 was taken for normal prostate and cancerous regions, and an excision of 5mm2 was taken for microenvironment due to the smaller area available for this region across slides. After the buffers have been prepared, proceed to sample excision. Begin by scraping away paraffin adjacent to the tissue area of interest with a single-edge disposable razor blade. Excess paraffin can form a thick layer on top of the liquid in subsequent steps, inhibiting digestion and mixing. Prepare a new 96-well PCR plate, then proceed with one sample at a time. Add 1.8 μL of 1X Lysis Buffer per mm2 of sample tissue to the well. Attach a fresh sterile tip to a single-channel hand pipet or prepare a sterile plastic toothpick. Use a fresh tip/toothpick for every sample. Excise the target area by scraping it off the glass slide with a disposable razor. Use a fresh razor for each sample. Transfer the excised tissue into the plate well. Use the pipet tip or toothpick to push the sample off the blade, into the well, and down to the bottom of the well. Static electricity may cause the tissue to fly off the transfer tool. This can be prevented by pre-wetting the transfer tool in lysis buffer by dipping it into the well. If using a pipette tip, dispense any liquid that may have entered the tip by capillary action into the well. Overlay sample with molecular biology grade mineral oil. Use 10 μL plus 1 μL per mm2 of tissue in the sample. For example, add 20 μL to a 10 mm2 tissue input. When all samples have been processed, visually inspect the plate to ensure there is tissue at the bottom of each assay well. Spin the plate at 1000 rcf for 2 minutes. Incubate the plate at 95°C for 5 minutes to melt the tissue, then cool it to 4°C for 5 minutes. Add 0.2 μL Protease Mix per mm2 of tissue to the aqueous layer at the bottom of each sample well. For example, add 2 μL for a 10 mm2 input. Mix the aqueous layer by trituration, taking care not to aspirate the tissue sample. Spin the plate at 1000 rcf for 2 minutes to bring the aqueous phase to the bottom of the well. This will also separate the oil/paraffin layer from the lysate. Incubate the plate at 37°C for 30 minutes for tissue lysis. After tissue lysis, visually inspect the wells. You may see tissue fragments in the aqueous layer. To homogenize the lysate, triturate until the lysate becomes uniform. Take care to avoid bubbles. Spin the plate at 1000 rcf for 2 minutes to separate layers. FFPE lysates can be used immediately or stored at -80°C for up to one year. The TempO-Seq assay utilizes a detector oligo (DO) pool specific to the RNA targets to be monitored. BioSpyder offers fixed content and custom DO pools to measure focused sets of transcripts, up to whole transcriptomes. In addition to FFPE tissues, the assay can utilize purified RNA, and cell lysates. Both positive and negative control samples can be included. The TempO-Seq assay does not require extraction or purification of RNA from FFPE samples. Instead, tissue is scraped from a slide into lysis buffer, melted, and lysed. This tissue lysate is directly added into the assay. RNA within the lysate is annealed to a matched pair of detector oligos, with each pair of DOs targeting a single transcript. The DO pool targets up to tens of thousands of RNA sequences, depending on the content of the assay. The DOs hybridize to sequences of the targeted RNA molecule such that their respective 3' and 5' ends are immediately adjacent. After DO annealing, excess oligos and mis-hybridized or weakly hybridized DOs are removed by nuclease digestion, and the annealed oligos are ligated to form amplifiable templates. The ligated DOs are amplified with one pair of primers (making each PCR reaction single-plex regardless of the number of genes measured). A different combination of sample index/tag sequences can be assigned to each sample. The PCR products are pooled together into a sequencing library, which is then purified and run on an Illumina sequencer to count the number of ligated detector oligo sequences per sample (ThermoFisher-compatible kit under development) . Because TempO-Seq includes an end-point PCR reaction, individual quantitation of the amplified products before library pooling is not needed. Very low concentration and negative control samples are expected to show low reads in the sequencing data. The resulting sequencing read counts from the pooled library can be analyzed to determine the abundance of each gene from each sample. Many types of further analysis are then possible; for example, changes in expression due to treatments or differential expression between samples can be determined. In TempO-Seq, each Detector Oligo consists of a sequence complementary to an mRNA target plus a universal (i.e. same for every targeted gene) primer binding site. They anneal in immediate juxtaposition to each other on the targeted RNA template such that they can be ligated together. Ligated detector oligos are PCR-amplified using a primer set (single-plex PCR reaction, with a single primer pair for each sample) that introduces both the adaptors required for sequencing and a sample-specific barcode. The barcode sequences flank the target sequence and are inserted appropriately into the standard Illumina adaptors to permit standard dual-index sequencing of the barcodes and deconvolution of sample-specific reads from the sequencing data using the standard Illumina software. All the PCR-amplified and barcoded samples are pooled into a single library for sequencing. Sequencing reads are demultiplexed using the standard sequencing instrument software for each sample using the barcodes to give a FASTQ file for each.
attributes.tag: ENA-FIRST-PUBLIC
attributes.value: 2023-01-29
attributes.tag: ENA-LAST-UPDATE
attributes.value: 2023-01-29
attributes.tag: External Id
attributes.value: SAMEA112354460
attributes.tag: INSDC center alias
attributes.value: DUNDEE
attributes.tag: INSDC center name
attributes.value: University of Dundee
attributes.tag: INSDC first public
attributes.value: 2023-01-29T00:19:35Z
attributes.tag: INSDC last update
attributes.value: 2023-01-29T00:19:35Z
attributes.tag: INSDC status
attributes.value: public
attributes.tag: Submitter Id
attributes.value: E-MTAB-12593:Sample 2_ME
attributes.tag: broker name
attributes.value: ArrayExpress
attributes.tag: common name
attributes.value: human
attributes.tag: disease
attributes.value: prostate cancer
attributes.tag: individual
attributes.value: individual 2
attributes.tag: organism part
attributes.value: prostate gland
attributes.tag: sample name
attributes.value: E-MTAB-12593:Sample 2_ME
attributes.tag: sampling site
attributes.value: microenvironment
attributes.tag: sex
attributes.value: male