Can tRNA and tRF/tiRNA be profiled in the same sequencing experiment?
tRNA (60-100 nt) and tRF/tiRNA (15-45 nt) have different size ranges, RNA pretreatment, cDNA synthesis, and library construction requirements. Currently we use enhanced hydro-tRNA-seq for tRNA, where full length mature tRNAs are first isolated by gel electrophoresis and then partially hydrolyzed to overcome strong tRNA structure and modification problems. This workflow is not compatible with tRF/tiRNA-seq.
For non-model organisms, can Arraystar provide tRNA/tRF/tiRNA sequencing?
If you can provide the transcriptome, tRNA, and genome reference databases for non-model species other than human, mouse, or rat, such as Plasmodium falciparum, we can provide sequencing and data analysis services. If no reference database is available, we may provide raw data-only sequencing for non-modeling organisms. Please inquire to confirm.
Is the rtStar™ tRNA Pretreatment Kit required for tRNA PCR Array?
For tRNA cDNA synthesis, the rtStar™ tRNA-optimized First-Strand cDNA Synthesis Kit (AS-FS-004) includes an RNA demethylation step that enzymatically removes m1A, m1G, and m3C. For tRF and tiRNA cDNA synthesis, the rtStar™ tRF and tiRNA Pretreatment Kit (AS-FS-005) prepares fragment termini for adapter ligation and removes m1A, m1G, and m3C.
How should I submit RNA for tRNA and small RNA sequencing?
Our sequencing systems have been specifically optimized for total RNA extracted using the TRIzol method. To avoid small RNA loss during purification, we recommend you submit total RNA, not isolated small RNAs. If you have isolated miRNA, technically we can do it, but we cannot assure the success of library preparation affected by small RNA extraction efficiency.
Should I send FFPE samples or extracted RNA?
The main problem with FFPE samples is that the RNA is typically degraded, so obtaining the highest yield and quality of RNA from de-paraffinization and extraction is essential for high quality profiling data. Arraystar has many years of experience extracting RNA from FFPE samples using optimized protocols, and we strongly recommend sending FFPE samples to us for extraction.
Which method do you recommend for total RNA extraction?
At Arraystar we use TRIzol Reagent for total RNA extraction from cells and tissues. If you have experience with total RNA isolation, use whichever method you are most comfortable with, such as Qiagen RNeasy kits. The key objective is high yield with minimal degradation and contamination from proteins, DNA, and organic solvents.
Why do I need biological replicates?
Biological replicates increase the probability that your array results are statistically significant. Genetic differences exist between individuals with the same apparent phenotype, even in inbred laboratory strains. Testing only one individual per group cannot show whether an observed difference is due to experimental conditions, so more than one individual per group is needed to confirm real differential expression.
How many biological replicates do you recommend?
We strongly recommend a minimum of three biological replicates per group for the most statistically significant data. With one sample per group you never know if that individual is an outlier, and with two you cannot tell which individual is the outlier. For mammalian tissues we prefer six biological replicates per group due to tissue heterogeneity.
Why use Small RNA Array instead of small RNA-seq?
Arraystar Small RNA Array uses one single labeling reaction to cover major small RNA classes, including miRNA, pre-miRNA, tRNA, tsRNA, and snoRNA, at once, whereas multiple sequencings would be required for biochemically different small RNA classes. Direct end labeling avoids complicated pretreatments and size selection, achieving higher sensitivity and accuracy with less sample.
How are fold changes calculated in microarray experiments?
We do not use raw signal intensities for fold changes; we only use normalized values. Normalized intensities are log2 transformed, and fold change is calculated by subtracting the normalized intensity of one group from another, then converting the log2 difference back to a linear value, shown in the FC (abs) column.
How do I pick differentially expressed transcripts to validate?
Researchers can validate microarray data in at least two ways: validate all differentially expressed transcripts on the array in an unbiased, high-throughput manner, or focus on transcripts associated with a biological function of interest using the GO and Pathway analysis. We recommend eliminating transcripts with extremely low raw signals and selecting larger fold changes with lower p values.
Does Arraystar provide a quote for my tRNA project?
Yes. Click the Request Quote button on any page to open the inquiry form, then share your project details, including sample type, species, and sample number. Our team will respond with a tailored quote, statement of work, and project timeline for the tRNA service or product you need.
Where can I find the full Arraystar FAQ?
This page distills the questions most relevant to tRNA, tRF and tiRNA research. The complete official Arraystar FAQ covers additional technologies, including lncRNA arrays, circRNA arrays, epitranscriptomic arrays, MeRIP-seq, and MeDIP-seq, and is available on the Arraystar website for full details.
Do you recommend biological or technical replicates?
We strongly recommend biological replicates, with a minimum of three per group and six preferred for mammalian tissues. Technical replicates are not needed because the arrays have been validated many times and contain control probes, so they would be a waste of money.
Can degraded or biofluid RNA be profiled?
Yes. Small RNA arrays and tRF/tiRNA workflows tolerate lower quality RNA, and tRNA and tRF/tiRNA are highly enriched in biofluids, making them suitable for serum and plasma studies. FFPE samples are best sent to Arraystar for optimized RNA extraction and quality control.
What sample amounts are required for tRNA services?
Recommended minimums are more than 1 µg of total RNA for tRF/tiRNA-seq, more than 2 µg for tRNA-seq, and more than 5 µg for PCR array projects. Supplying twice the recommended minimum amount helps avoid project delays due to QC.
How do I start a service project?
Send your project basics by email to the customer specialist or to support@arraystar.com, including sample number, species, sample conditions, and experimental design. After initial consultation you will receive a quotation, statement of work, and service contract for your review and approval.
