RNA-seq Week 2018

RNA-seq Week 2018 – valuable insights and Q&A from the webinar series

Life science research is constantly on the edge of new technology developments. This trend is especially apparent in advanced transcriptomics and gene expression research. In December 2018, we hosted “RNA-seq Week”, where we presented five different webinars addressing the complete gene expression workflow, starting from sample preparation and ending with bioinformatics analysis. It also included a special discussion of products offering Locked Nucleic Acid (LNA) chemistry.  

We captured a lot of interesting questions from our webinar attendees. Read our compilation of the top 10 questions from researchers who attended these webinars, along with insightful answers from the QIAGEN experts.  

To listen to the recordings of these webinars, as well as post-webinar discussions, click on the links below.

“Sequence what matters – one-step rapid removal of rRNA and/or globin mRNA using QIAseq FastSelect” presented by Jonathan Shaffer, Ph.D., M.B.A., Associate Director, NGS Assay Technologies, QIAGEN 

Recorded webinar session – click here 

Whole transcriptome NGS enables the characterization of both coding mRNAs and long noncoding RNAs (lncRNAs) from biological samples, including FFPE samples. However, before ultra-sensitive RNA-seq can be performed on FFPE samples or other sample types, cytoplasmic and mitochondrial rRNA should be removed to increase sensitivity and decrease the cost per sample. To deplete rRNA, various methodologies exist, but unfortunately they are not efficient enough. In this webinar, Dr. Jonathan Shaffer presented the new QIAseq FastSelect RNA Removal Kits, which are based on a novel, one-step rRNA depletion technology and discussed how these kits can simplify and speed up rRNA removal in RNA-seq applications. 

Post-webinar Q&A: 

1. During RNA-seq sample prep, is ribosomal RNA (rRNA) depletion always recommended –under what circumstances would it not be necessary? 

I typically always recommend rRNA depletion, as opposed to mRNA selection, because rRNA depletion gives you the broadest potential for discovery. With rRNA depletion, mRNAs and long noncoding RNAs remain, giving you the greatest chance for discovery. If you only select for mRNAs, you are removing the chance for discovery of long noncoding RNA signatures.

2. Is it possible to use the QIAseq FastSelect RNA Depletion Kit for rRNA depletion from mammalian gut samples that additionally possess bacteria which are of interest? 

Currently, QIAseq FastSelect really isn’t suitable for most bacterial applications. The method by which QIAseq FastSelect works is largely species-specific, while many bacterial applications require a “pan-depletion” due to the presence of multiple bacterial species. Use of the current kit version with a mixed mammalian/bacterial RNA sample would result in removal of the mammalian rRNA but not the bacterial rRNA. QIAGEN is in the process of evaluating different methodologies for a bacterial-depletion approach, and we hope to be able to add such a bacterial rRNA depletion kit version to our portfolio in the future. 

“QIAseq UPX Kits for 3’ RNA-seq with low-input or single-cell samples” presented by Samuel Rulli, Ph.D., Senior Global Product Manager, RNA Profiling and Assay Technologies, QIAGEN

Recorded webinar session – click here

In this webinar session, Dr. Samuel Rulli explained the advantages of using QIAGEN’s QIAseq UPX 3’ Transcriptome Kits for library preparation and GeneGlobe Data Analysis Center to overcome challenges related to using the 3’ next-generation sequencing (NGS) method for gene expression analysis. QIAGEN’s QIAseq UPX 3’ Kits utilize unique molecular indices (UMIs) to increase the accuracy of RNA-seq, as well as LNA-enhanced chemistry for superior sensitivity. The webinar provided an overview of these technologies, in addition to a simplified library preparation strategy to complete the workflow. Dr. Rulli also focused on QIAGEN’s data analysis solution, which allows you to go from FASTQ file to fine differential fold change or single-cell analysis.  

Post-webinar Q&A: 

1. Because miRNA binds to 3’ non-translated ends of RNA, could this binding interfere with 3’ RNA sequencing? 

We perform the RT reaction at 42oC which should be adequate to allow miRNA to disassociate from any RNA transcripts. The reverse transcriptase enzyme we use has been modified to have greater stability at 42 oC and increased strand displacement activity as compared to the wildtype enzyme.  

2. My research focuses on mammalian poly-A tail sequences analysis, however, my samples also contain a lot of bacteria and bacterial RNA. Will the presence of bacterial RNA affect capture of poly-A targets by the QIAseq UPX 3’ Transcriptome Kit? 

The presence of bacterial RNA should not affect the performance of the QIAseq UPX 3’ Kits since we use the poly-A tail to generate cDNA with our oligo-dt primer. 

“Sample to Insight: Everything you need to know about RNA sequencing”, presented by Dr. Jonathan Shaffer, Ph.D., M.B.A., Associate Director, NGS Assay Technologies, QIAGEN 

Recorded webinar session – click here 

RNA sequencing unlocks the mysteries hidden in the transcriptome. Whether the experimental goal is gene expression analysis, gene fusion analysis, SNP analysis or miRNA expression analysis, achieving high-performance library preparation can prove essential to maximizing research insights.  

In this webinar, Dr. Jonathan Shaffer explained the best practices, considerations and tips for library prep, and provided valuable advice for sample prep, sample QC, sequencing, data analysis and data interpretation to achieve the greatest RNA-seq outcomes.

Post-webinar Q&A: 

1. Once I obtain my differential gene expression data, what would be the best strategy for me to then perform functional pathway mapping? 

The best approach for this is to use Ingenuity Pathways Analysis (IPA). With IPA, you can understand the significance of your data with respect to biological systems, both in terms of what is already published and what is unknown. 

2. Are there instances where RNA fragmentation is not necessary as part of library prep?  If so, when could I omit this step?

RNA fragmentation is not required when samples are already degraded, for example FFPE samples. The integrity of FFPE samples can be assessed. Based on an integrity score, the sample may either need less fragmentation or no fragmentation at all. 

“miRNA qPCR for challenging samples”, presented by Michael Bussmann, Ph.D., Senior Global Product Manager, PCR Systems and Assays, QIAGEN  

Recorded webinar session – click here

This webinar, led by Dr. Michael Bussmann, discusses the important considerations of miRNA analysis when working with challenging sample types. Use of Locked Nucleic Acid (LNA) technology is proving instrumental for success in these situations. Dr. Bussmann explained how LNA technology, an integral part of the miRCURY LNA miRNA PCR System, deals with challenging clinical biofluid samples and discussed the importance of quality control and data analysis in PCR. 

Post-webinar Q&A: 

1. You told us that pre-amplification is not required, but if I refer to the QIAGEN guidelines for miRNA assays, they do recommend pre-amplification when dealing with serum or plasma. Can you explain why? 

These  recommendations (miRNA guidelines) are true for the miScript system, but not for the new miRCURY LNA System. Using the LNA-enhanced PCR system, pre-amplification is not needed, even from samples such as serum/plasma. QIAGEN is currently preparing a new manual on miRNA guidelines, which should be available later this year.
 

2. For a single target assay, do you need perform miRNA enrichment, or would total RNA isolation work?

For a single miRNA PCR Assay, as well as PCR panels, total RNA works. 

“Take your RNA research to the next level with LNA technologies”, presented by Roman Kurek, Ph.D., Senior Customs Solutions Manager, Genomics, QIAGEN

Recorded webinar session – click here 

QIAGEN has over twenty years of experience in LNA oligo design to enhance qPCR technology, in situ hybridization, functional analysis of RNA in cells and animals, miRNA and RNA sequencing and much more. LNA oligos bind with much higher affinity and specificity to RNA targets than standard DNA and RNA oligos. This enables specific and sensitive detection of small RNAs and discrimination between highly similar sequences. In this webinar, Dr. Roman Kurek presented the unique features and benefits of LNA technology and discussed how your miRNA qPCR experiments and your functional analyses of RNA will benefit from its use. 

Post-webinar Q&A: 

1. You have shown at the very beginning of your presentation, how you can use LNA to make oligos shorter. How short can you actually go? 

You can, in most cases, go down to 15–16 nt. Rarely, you can go even shorter and there are some publications with so-called tinymers (down to eight nucleotides in length). These are special cases and LNA oligonucleotides that short very often show nonspecific side effects due to having a number of LNA modifications placed so close to each other, which makes the oligonucleotide very sticky and nonspecific. It is furthermore very likely that there are a significant number of perfect matches in a eukaryotic genome. 

2. What’s the chemical difference between an miRNA inhibitor and a target site blocker?

LNA miRNA inhibitors and LNA target side blockers are chemically identical. Both are LNA-enhanced oligonucleotides with a sufficiently short LNA spacing to completely abolish RNase H activity. The difference between both is their mode of action: An LNA miRNA inhibitor binds to its mature miRNA target and prevents recruitment and guidance of all targeted mRNAs into the RISC complex. An LNA target site blocker blocks the interaction of an miRNA with one defined mRNA target by masking the miRNA binding site in the 3’-UTR of the specific mRNA. So in contrast to an LNA miRNA inhibitor (which is designed against an miRNA), an LNA target site blocker is designed against the 3’-UTR of an mRNA.

Summary

The “RNA-seq Week 2018” webinar series presented a lot of valuable information about QIAGEN technologies for gene expression. We hope the insights provided by the webinar series, as well as the Q&A session help to take your research to the next level!

If you want to know more about our solutions, please click on the links below to dive into the details.

NGS in gene expression analysis:

• QIAseq Stranded RNA Library Kits
• QIAseq Targeted RNA Panels
• QIAseq UPX 3’ Transcriptome Kits
• QIAseq UPX 3’ Targeted RNA Panels
• QIAseq miRNA Library Kit
• QIAseq FastSelect RNA Removal Kits

 

LNA enhanced miRNA qPCR: 

• miRCURY LNA miRNA PCR Assays