QIAseq FastSelect RNA Removal is a one-step rRNA and/or globin mRNA depletion solution. Simply add QIAseq FastSelect reagent (rRNA Removal and/or Globin Removal) to RNA sample, perform fragmentation (if required), stepwise cool the reaction from 75°C to 25°C for 20 minutes and then complete the remaining library preparation steps. QIAseq FastSelect works with or without RNA fragmentation, providing the flexibility to use FFPE or degraded RNA samples, or high-quality RNA as part of a standard RNA-seq library construction workflow.
QIAseq FastSelect has been tested with the QIAseq Stranded Total RNA Lib Kit (QIAGEN), TruSeq Stranded (Illumina), NEBNext Ultra II Directional (New England Biolabs) and KAPA RNA HyperPrep (KAPA Biosystems) library preparation kits. Specific requirements for fragmentation, as well as the duration of the fragmentation process, depend on the library preparation kit and sample type. Fragmentation is not required for QIAseq FastSelect performance.
Stranded transcriptome libraries were prepared from 100 ng aliquots of Universal Human Reference RNA (UHRR) (Agilent) using the QIAseq Stranded Total RNA Lib Kit. UHRR samples were either: 1. Not treated, 2. Poly-A enriched, 3. rRNA-depleted with Ribo-Zero Gold or 4. rRNA-depleted using QIAseq FastSelect. Poly-A enrichment and Ribo-Zero Gold rRNA depletion were performed according to the manufacturers’ instructions. For QIAseq FastSelect rRNA depletion, during the QIAseq library preparation, rRNA removal reagent was combined with each RNA sample and 5x RT Buffer. Next, the samples were heated (fragmented) at 95°C for 15 min and then stepwise cooled to 25°C for 20 minutes. Afterwards, the remaining library preparation steps were performed. Sequencing was performed on a NextSeq 550 (Illumina) and data analysis was performed using CLC Biomedical Workbench.
A and B QIAseq FastSelect results in highly efficient removal of rRNA to levels observed with Poly-A enrichment. Average % rRNA remaining is provided in A and the individual replicates are plotted in B.
Stranded transcriptome libraries were prepared from 100 ng aliquots of Universal Human Reference RNA (UHRR) (Agilent) using the QIAseq Stranded Total RNA Lib Kit. UHRR samples were either: 1. Not treated, 2. Poly-A enriched, 3. rRNA-depleted with Ribo-Zero Gold or 4. rRNA-depleted using QIAseq FastSelect. Poly-A enrichment and Ribo-Zero Gold rRNA depletion were performed according to the manufacturers’ instructions. For QIAseq FastSelect rRNA depletion, during the QIAseq library preparation, rRNA removal reagent was combined with each RNA sample and 5x RT Buffer. Next, the samples were heated (fragmented) at 95°C for 15 min and then stepwise cooled to 25°C for 20 minutes. Afterwards, the remaining library preparation steps were performed. Sequencing was performed on a NextSeq 550 (Illumina) and data analysis was performed using CLC Biomedical Workbench.
A When QIAseq FastSelect is used for rRNA depletion, gene expression results are highly similar to results observed when using Ribo-Zero Gold (log2 normalized gene expression), demonstrating the efficacy of QIAseq FastSelect. B Gene expression results are highly similar between QIAseq FastSelect and untreated samples (no treatment), suggesting QIAseq FastSelect does not perturb the natural expression profiles of samples (targets with greater than 10 reads in both sample sets [n=3 each] have been plotted). C Gene expression profiles from Poly-A enriched samples are very different from untreated samples (no treatment), suggesting Poly-A enrichment perturbs the natural expression profile of samples (targets with greater than 10 reads in both sample sets [n=3] have been plotted).
Stranded transcriptome libraries were prepared from 100 ng aliquots of the SEQC reference samples using the QIAseq Stranded Total RNA Lib Kit (Universal Human Reference RNA = UHRR [Agilent] and Human Brain Reference RNA = HBRR [Thermo Scientific]). QIAseq FastSelect was used for rRNA depletion. During the QIAseq library preparation, rRNA removal reagent was combined with each RNA sample and 5x RT Buffer. Next, the samples were heated (fragmented) at 95°C for 15 min and then stepwise cooled to 25°C for 20 minutes. Afterwards, the remaining library preparation steps were performed. Sequencing was performed on a NextSeq 550 (Illumina) and data analysis was performed using CLC Biomedical Workbench.
QIAseq FastSelect results in highly efficient removal of rRNA from the SEQC samples. Average % rRNA remaining is provided in A and the individual replicates are plotted in B.
Stranded transcriptome libraries were prepared from 100 ng aliquots of the SEQC reference samples using the QIAseq Stranded Total RNA Lib Kit (Universal Human Reference RNA = UHRR [Agilent] and Human Brain Reference RNA = HBRR [Thermo Scientific]). QIAseq FastSelect was used for rRNA depletion. During the QIAseq library preparation, rRNA removal reagent was combined with each RNA sample and 5x RT Buffer. Next, the samples were heated (fragmented) at 95°C for 15 min and then stepwise cooled to 25°C for 20 minutes. Afterwards, the remaining library preparation steps were performed. Sequencing was performed on a NextSeq 550 (Illumina) and data analysis was performed using CLC Biomedical Workbench.
Three replicates were performed for each sample and gene expression results are highly reproducible (log2 normalized gene expression) when QIAseq FastSelect is used for rRNA depletion.
Stranded transcriptome libraries were prepared from 100 ng aliquots of the SEQC reference samples using the QIAseq Stranded Total RNA Lib Kit (Universal Human Reference RNA = UHRR [Agilent] and Human Brain Reference RNA = HBRR [Thermo Scientific]). QIAseq FastSelect was used for rRNA depletion. During the QIAseq library preparation, rRNA removal reagent was combined with each RNA sample and 5x RT Buffer. Next, the samples were heated (fragmented) at 95°C for 15 min and then stepwise cooled to 25°C for 20 minutes. Afterwards, the remaining library preparation steps were performed. Sequencing was performed on a NextSeq 550 (Illumina) and data analysis was performed using CLC Biomedical Workbench.
Principal component analysis (PCA) of expression profiles demonstrate that the replicate samples cluster as expected with each other and the four reference samples cluster as expected in relation to one another.
Stranded transcriptome libraries were prepared from 100 ng aliquots of the SEQC reference samples using the QIAseq Stranded Total RNA Lib Kit (Universal Human Reference RNA = UHRR [Agilent] and Human Brain Reference RNA = HBRR [Thermo Scientific]). QIAseq FastSelect was used for rRNA depletion. During the QIAseq library preparation, rRNA removal reagent was combined with each RNA sample and 5x RT Buffer. Next, the samples were heated (fragmented) at 95°C for 15 min and then stepwise cooled to 25°C for 20 minutes. Afterwards, the remaining library preparation steps were performed. Sequencing was performed on a NextSeq 550 (Illumina) and data analysis was performed using CLC Biomedical Workbench.
Venn diagram of differentially expressed genes demonstrates unique and overlapping expression profiles across the samples. The Venn diagram shows the number of genes with ≥2-fold change when FDR p-value is ≤0.05.
Total RNA was isolated from 5 µm normal and cancer lung FFPE sections using the miRNeasy FFPE Kit. Stranded transcriptome libraries were subsequently prepared from 100 ng aliquots using the QIAseq Stranded Total RNA Lib Kit. For rRNA depletion, QIAseq FastSelect or Ribo-Zero Gold was used. Sequencing was performed on a NextSeq 550 (Illumina) and data analysis was performed using CLC Biomedical Workbench. QIAseq FastSelect: During the QIAseq library preparation, rRNA removal reagent was combined with each RNA sample and 5x RT Buffer, heated (fragmented) to 75°C for 2 min and stepwise cooled to 25°C for 20 minutes. Afterwards, the remaining library preparation steps were performed.
Ribo-Zero Gold: Prior to QIAseq library preparation, rRNA was removed from each RNA sample using Ribo-Zero Gold, which required over 2 hours to complete. Afterwards, QIAseq RNA library preparation was performed.
A Average % rRNA remaining. B Identical colors represent the same sample and replicates. QIAseq FastSelect results in highly efficient removal of rRNA from fragmented samples. Average % rRNA remaining is provided in A and plotted in B. Ribo-Zero Gold required substantially more amplification cycles than the QIAseq FastSelect libraries, suggesting some sample may be lost with Ribo-Zero Gold.
Total RNA was isolated from 5 µm normal and cancer lung FFPE sections using the miRNeasy FFPE Kit. Stranded transcriptome libraries were subsequently prepared from 100 ng aliquots using the QIAseq Stranded Total RNA Lib Kit. For rRNA depletion, QIAseq FastSelect or Ribo-Zero Gold was used. Sequencing was performed on a NextSeq 550 (Illumina) and data analysis was performed using CLC Biomedical Workbench. QIAseq FastSelect: During the QIAseq library preparation, rRNA removal reagent was combined with each RNA sample and 5x RT Buffer, heated (fragmented) to 75°C for 2 min and stepwise cooled to 25°C for 20 minutes. Afterwards, the remaining library preparation steps were performed.
Ribo-Zero Gold: Prior to QIAseq library preparation, rRNA was removed from each RNA sample using Ribo-Zero Gold, which required over 2 hours to complete. Afterwards, QIAseq RNA library preparation was performed.
Performing rRNA depletion using QIAseq FastSelect achieved highly reproducible gene expression results (log2 normalized gene expression) for A normal FFPE RNA and B cancer FFPE RNA. Two replicates were performed for each sample type.
Total RNA was isolated from FFPE reference standards (Fusion RNA Multiplex Positive and Negative Controls [Horizon™]) using the miRNeasy FFPE Kit. Stranded transcriptome libraries were subsequently prepared from 100 ng aliquots using the QIAseq Stranded Total RNA Lib Kit. For rRNA depletion, QIAseq FastSelect or Ribo-Zero Gold was used. Sequencing was performed on a NextSeq 550 (Illumina) and data analysis was performed using CLC Biomedical Workbench. QIAseq FastSelect: During the QIAseq library preparation, rRNA removal reagent was combined with each RNA sample and 5x RT Buffer. Next, the samples were heated (fragmented) at 95°C for 15 min and then stepwise cooled to 25°C for 20 minutes. Afterwards, the remaining library preparation steps were performed.
Ribo-Zero Gold: Prior to QIAseq library preparation, rRNA was removed from each RNA sample using Ribo-Zero Gold, which required over 2 hours to complete. Afterwards, QIAseq RNA library preparation was performed.
A Average % rRNA remaining. B Identical colors represent the same sample and replicates. Average % rRNA remaining is provided in A and the individual replicates are plotted in B. QIAseq FastSelect demonstrated highly efficient removal of rRNA from FFPE standards. Ribo-Zero Gold required substantially more amplification cycles than the QIAseq FastSelect libraries, suggesting some sample may be lost with Ribo-Zero Gold.
Total RNA was isolated from FFPE reference standards (Fusion RNA Multiplex Positive and Negative Controls [Horizon]) using the miRNeasy FFPE Kit. Stranded transcriptome libraries were subsequently prepared from 100 ng aliquots using the QIAseq Stranded Total RNA Lib Kit. For rRNA depletion, QIAseq FastSelect or Ribo-Zero Gold was used. Sequencing was performed on a NextSeq 550 (Illumina) and data analysis was performed using CLC Biomedical Workbench. QIAseq FastSelect: During the QIAseq library preparation, rRNA removal reagent was combined with each RNA sample and 5x RT Buffer. Next, the samples were heated (fragmented) at 75°C for 2 min and then stepwise cooled to 25°C for 20 minutes. Afterwards, the remaining library preparation steps were performed.
Ribo-Zero Gold: Prior to QIAseq library preparation, rRNA was removed from each RNA sample using Ribo-Zero Gold, which required over 2 hours to complete. Afterwards, QIAseq RNA library preparation was performed.
Performing rRNA depletion using QIAseq FastSelect achieved highly reproducible gene expression results (log2 normalized gene expression) for A Fusion RNA Multiplex Positive FFPE and B Fusion RNA Multiplex Negative FFPE. Two replicates were performed for each sample type.
Stranded transcriptome libraries were prepared from 100 ng aliquots of human tissue RNA samples using the QIAseq Stranded Total RNA Lib Kit. QIAseq FastSelect was used for rRNA depletion. During the QIAseq library preparation, rRNA removal reagent was combined with each RNA sample and 5x RT Buffer. Next, the samples were heated (fragmented) at 95°C for 15 min and then stepwise cooled to 25°C for 20 minutes. Afterwards, the remaining library preparation steps were performed. Sequencing was performed on a NextSeq 550 (Illumina) and data analysis was performed using CLC Biomedical Workbench.
A Average % rRNA remaining. B Tissue replicates (n=3). QIAseq FastSelect results in highly efficient removal of rRNA from RNA samples extracted from various human tissues. Average % rRNA remaining is provided in A and the individual tissue replicates are plotted in B.
Stranded transcriptome libraries were prepared from 100 ng aliquots of human tissue RNA samples using the QIAseq Stranded Total RNA Lib Kit. QIAseq FastSelect was used for rRNA depletion. During the QIAseq library preparation, rRNA removal reagent was combined with each RNA sample and 5x RT Buffer. Next, the samples were heated (fragmented) at 95°C for 15 min and then stepwise cooled to 25°C for 20 minutes. Afterwards, the remaining library preparation steps were performed. Sequencing was performed on a NextSeq 550 (Illumina) and data analysis was performed using CLC Biomedical Workbench.
Performing rRNA depletion using QIAseq FastSelect achieved highly reproducible gene expression results (log2 normalized gene expression) for RNA samples extracted from various human tissues. Three replicates were performed for each sample.
Stranded transcriptome libraries were prepared from 100 ng and 1µg aliquots of HeLa S3 and HCT 116 total RNA samples using the QIAseq Stranded Total RNA Lib Kit. QIAseq FastSelect was used for rRNA depletion. During the QIAseq library preparation, rRNA removal reagent was combined with each RNA sample and 5x RT Buffer. Next, the samples were heated (fragmented) at 95°C for 15 min and then stepwise cooled to 25°C for 20 minutes. Afterwards, the remaining library preparation steps were performed. Sequencing was performed on a MiSeq (Illumina) and data analysis was performed using CLC Biomedical Workbench.
A Average % rRNA remaining. B Blue = QIAseq FastSelect and red = no treatment. QIAseq FastSelect results in highly efficient removal of rRNA compared to untreated samples. Average % rRNA remaining is provided in A and plotted in B.
Stranded transcriptome libraries were prepared from 100 ng and 1µg aliquots of A HeLa S3 and B HCT 116 total RNA samples using the QIAseq Stranded Total RNA Lib Kit. QIAseq FastSelect was used for rRNA depletion. During the QIAseq library preparation, rRNA removal reagent was combined with each RNA sample and 5x RT Buffer. Next, the samples were heated (fragmented) at 95°C for 15 min and then stepwise cooled to 25°C for 20 minutes. Afterwards, the remaining library preparation steps were performed. Sequencing was performed on a MiSeq (Illumina) and data analysis was performed using CLC Biomedical Workbench.
Performing rRNA depletion using QIAseq FastSelect achieved highly reproducible gene expression results (log2 normalized gene expression) for RNA samples extracted from cell lines when comparing 100 ng (X-axis) to 1 µg (Y-axis). Two replicates were performed for each sample.
Stranded transcriptome libraries were prepared from 100 ng aliquots of Universal Human Reference RNA (UHRR) (Agilent) using the QIAseq Stranded Total RNA Lib Kit. For rRNA depletion, two different lots of QIAseq FastSelect were used. During the QIAseq library preparation, rRNA removal reagent was combined with each RNA sample and 5x RT Buffer. Next, the samples were heated (fragmented) at 95°C for 15 min and then stepwise cooled to 25°C for 20 minutes. Afterwards, the remaining library preparation steps were performed. Sequencing was performed on a MiSeq (Illumina) and data analysis was performed using CLC Biomedical Workbench.
A Average % rRNA remaining and % protein coding reads. B Blue = QIAseq FastSelect lot 1 and red = QIAseq FastSelect lot 2. QIAseq FastSelect results in highly efficient removal of rRNA regardless of kit lot. Average % rRNA remaining is provided in A and plotted in B.
Stranded transcriptome libraries were prepared from 100 ng aliquots of Universal Human Reference RNA (UHRR) (Agilent) using the QIAseq Stranded Total RNA Lib Kit. For rRNA depletion, two different lots of QIAseq FastSelect were used. During the QIAseq library preparation, rRNA removal reagent was combined with each RNA sample and 5x RT Buffer. Next, the samples were heated (fragmented) at 95°C for 15 min and then stepwise cooled to 25°C for 20 minutes. Afterwards, the remaining library preparation steps were performed. Sequencing was performed on a MiSeq (Illumina) and data analysis was performed using CLC Biomedical Workbench.
Performing rRNA depletion using QIAseq FastSelect achieved highly reproducible gene expression results (log2 normalized gene expression) when using different kit lots. Three replicates were performed for each sample.
Stranded transcriptome libraries were prepared from 100 ng and 1 µg aliquots of Universal Human Reference RNA (UHRR) (Agilent) using the Illumina TruSeq Stranded mRNA Library Prep kit (mRNA enrichment was not performed). For rRNA depletion, QIAseq FastSelect was used. During the TruSeq library preparation, rRNA removal reagent was combined with each RNA sample and FPF (from the TruSeq kit). Next, the samples were heated (fragmented) at 94°C for 8 min and then stepwise cooled to 25°C for 20 minutes. Afterwards, the remaining library preparation steps were performed according to the TruSeq recommendations. Sequencing was performed on a NextSeq 550 (Illumina) and data analysis was performed using CLC Biomedical Workbench.
A Average % rRNA remaining and % protein coding reads. B rRNA remaining is shown when no depletion is performed (no treatment) and when depletion is performed using QIAseq FastSelect (blue = 100 ng and red = 1 µg). QIAseq FastSelect results in highly efficient removal of rRNA. Average % rRNA remaining is provided in A and plotted in B.
Stranded transcriptome libraries were prepared from A 100 ng and B 1 µg aliquots of Universal Human Reference RNA (UHRR) (Agilent) using the Illumina TruSeq Stranded mRNA Library Prep kit (mRNA enrichment was not performed). For rRNA depletion, QIAseq FastSelect was used. During the TruSeq library preparation, rRNA removal reagent was combined with each RNA sample and FPF (from the TruSeq kit). Next, the samples were heated (fragmented) at 94°C for 8 min and then stepwise cooled to 25°C for 20 minutes. Afterwards, the remaining library preparation steps were performed according to the TruSeq recommendations. Sequencing was performed on a NextSeq 550 (Illumina) and data analysis was performed using CLC Biomedical Workbench.
Performing rRNA depletion using QIAseq FastSelect achieved highly reproducible gene expression results (log2 normalized gene expression). Two replicates were performed for each sample.
Stranded transcriptome libraries were prepared from 5 ng and 1 µg aliquots of Universal Human Reference RNA (UHRR) (Agilent) using the NEBNext Ultra II Directional RNA Library Prep kit (New England Biolabs). For rRNA depletion, QIAseq FastSelect was used. During the NEBNext library preparation, rRNA removal reagent was combined with each RNA sample, 5x NEBNext First Strand Synthesis Reaction Buffer (from the NEBNext kit) and random primers (from the NEBNext kit). Next, the samples were heated (fragmented) at 94°C for 15 min and then stepwise cooled to 25°C for 20 minutes. Afterwards, the remaining library preparation steps were performed according to the NEBNext recommendations. Sequencing was performed on a NextSeq 550 (Illumina) and data analysis was performed using CLC Biomedical Workbench.
A Average % rRNA remaining and % protein coding reads. B rRNA remaining is shown when no depletion is performed (no treatment) and when depletion is performed using QIAseq FastSelect (blue = 5 ng and red = 1 µg). QIAseq FastSelect results in highly efficient removal of rRNA. Average % rRNA remaining is provided in A and plotted in B.
Stranded transcriptome libraries were prepared from A 5 ng and B 1 µg aliquots of Universal Human Reference RNA (UHRR) (Agilent) using the NEBNext Ultra II Directional RNA Library Prep kit (New England Biolabs). For rRNA depletion, QIAseq FastSelect was used. During the NEBNext library preparation, rRNA removal reagent was combined with each RNA sample, 5x NEBNext First Strand Synthesis Reaction Buffer (from the NEBNext kit) and random primers (from the NEBNext kit). Next, the samples were heated (fragmented) at 94°C for 15 min and then stepwise cooled to 25°C for 20 minutes. Afterwards, the remaining library preparation steps were performed according to the NEBNext recommendations. Sequencing was performed on a NextSeq 550 (Illumina) and data analysis was performed using CLC Biomedical Workbench.
Performing rRNA depletion using QIAseq FastSelect achieved highly reproducible gene expression results (log2 normalized gene expression). Two replicates were performed for each sample.
Stranded transcriptome libraries were prepared from 25 ng and 1 µg aliquots of Universal Human Reference RNA (UHRR) (Agilent) using the KAPA RNA HyperPrep Kit (KAPA Biosystems). For rRNA depletion, QIAseq FastSelect was used. During the KAPA RNA HyperPrep workflow, rRNA removal reagent was combined with each RNA sample and Fragment, Primer and 2x Elute Buffer (from the KAPA RNA HyperPrep kit). Next, the samples were heated (fragmented) at 94°C for 8 min and then stepwise cooled to 25°C for 20 minutes. Afterwards, the remaining library preparation steps were performed according to the KAPA RNA HyperPrep recommendations. Sequencing was performed on a NextSeq 550 (Illumina) and data analysis was performed using CLC Biomedical Workbench.
A Average % rRNA remaining and % protein coding reads. B rRNA remaining is shown when depletion is performed using QIAseq FastSelect (blue = 25 ng and red = 1 µg). QIAseq FastSelect results in highly efficient removal of rRNA. Average % rRNA remaining is provided in A and plotted in B.
Stranded transcriptome libraries were prepared from A 25 ng and B 1 µg aliquots of Universal Human Reference RNA (UHRR) (Agilent) using the KAPA RNA HyperPrep Kit (KAPA Biosystems). For rRNA depletion, QIAseq FastSelect was used. During the KAPA RNA HyperPrep workflow, rRNA removal reagent was combined with each RNA sample and Fragment, Primer and 2x Elute Buffer (from the KAPA RNA HyperPrep kit). Next, the samples were heated (fragmented) at 94°C for 8 min and then stepwise cooled to 25°C for 20 minutes. Afterwards, the remaining library preparation steps were performed according to the KAPA RNA HyperPrep recommendations. Sequencing was performed on a NextSeq 550 (Illumina) and data analysis was performed using CLC Biomedical Workbench.
Performing rRNA depletion using QIAseq FastSelect achieved highly reproducible gene expression results (log2 normalized gene expression). Two replicates were performed for each sample.