More than Meets the Eye. Next Generation RNA Extraction

Transcription

1 More than Meets the Eye. Next Generation RNA Extraction

2 Discover the World of RNA There are solutions for every scientist s problems, no matter what the application is. provides kits for a variety of biological samples including cultured cells, solid tissues, FFPE, whole blood, plasma, serum, urine, viruses, insects, microbes, yeast, plants, stool, soil, and swabs. The only limiting factor is your imagination. Every kit is specially designed to address unique sample types to ensure the highest quality RNA is ready for all sensitive downstream applications including Next-Generation Sequencing, RT-qPCR, arrays, hybridization, etc. Highly Cited CRISPR-Cas9 Cpf1 is a Single RNA-Guided Endonuclease of a Class 2 CRISPR-Cas System (Zetsche B et al. 215) Direct-zol RNA facilitated discovery of a novel CRISPR endonuclease, Cpf1, allowing fine-tuning and increased flexibility for genome editing. Single Cell Analysis Single-cell RNA-Seq resolves cellular complexity in sensory organs from the neonatal inner ear (Burns JC et al. 215) Quick-RNA was used to extract RNA from FACS-sorted cells for single-cell RNA-seq and transcriptomics to analyze the developmental process of the ear. Next-Gen Sequencing Total RNA-seq to identify pharmacological effects on specific stages of mrna synthesis (Boswell SA et al. 217) RNA Clean & Concentrator was used to clean-up a RNA-Seq library prepared for downstream Illumina sequencing to study transcription and pre-mrna processing.

3 RNA Solutions for any Application Samples in TRIzol Cells Enzymatic Reactions Tissues FFPE Sample Input Biological Fluids Infectious Material Environmental (bacteria, fungi, plant, soil) Insect Vectors RNA from any Sample in TRIzol : Direct-zol RNA Kits RNA from any Cells & Tissue: Quick-RNA Kits DNA/RNA Co-Purification: Quick-DNA/RNA Kits RNA Clean-up: RNA Clean & Concentrator Kits Viral DNA/RNA Extraction: Quick-DNA/RNA Viral Kits Pathogen DNA/RNA Extraction: Quick-DNA/RNA Pathogen Kits Environmental RNA: Environmental RNA Kits Blood and Biological Fluids RNA: Quick-RNA Blood Kits Cell-free RNA: Quick-cfRNA Kits FFPE RNA Extraction: Quick-DNA/RNA FFPE Kit RNA Isolation Products

4 GET IT DIRECT TRIzol In. RNA Out. Direct-zol RNA Kits (Samples in TRIzol : cells, tissues, biological fluids) Easy Handling: No phase separation or precipitation steps. NGS-Ready: Ultra-pure RNA without phenol carryover. No DNA contamination (DNase I included). Non-Biased: Complete RNA recovery without mirna loss. 2

5 NGS-Ready RNA from TRIzol in 7 Minutes [kb] 1 Direct-zol Genomic DNA Contamination 1 [nt] Small RNAs Bind RNA directly to a spin-column 4 2 High-quality, intact, small and large RNA are efficiently recovered using a Direct-zol RNA kit compared to using a kit. RNA is DNA-free and ready for all downstream applications, including NGS. Non-Biased mirna Recovery Highest Yields mirna-seq 2837 overlapped mirna: r 2 =.976 1, Cells 3 Tissue (liver) 8, Unbiased Method (log 1 ) microrna (pg) 6, 4, 2, microrna (µg) 2 1 Direct-zol (log 1 ) Direct-zol Conventional Method Direct-zol Conventional Method RNA purified from TRIzol using Direct-zol RNA compared to an unbiased method (mirvana, Ambion). Data is highly correlated (2837 overlapped mirna: r 2 =.976). Analysis was performed using mirna-seq (MiSeq, Illumina). Direct-zol RNA kits recovered ~4-fold more mirna (<4 nt) than conventional methods. mirna purified from cells and tissue were quantified using Bioanalyzer small RNA chip. Product Cat. No. Size Binding Capacity Minimum Elution (Animal) Cells Tissue Direct-zol RNA Miniprep Plus Kit R27, R271* R272, R273* 1 µg 5 µl mg Direct-zol RNA Miniprep Kit R25, R251* R252, R253* 5 µg µl 5 x 1 6 mg Direct-zol RNA Microprep Kit R26, R261* R262, R263* 1 µg 6 µl mg Direct-zol -96 RNA Kit R254, R255* R256, R257* 1 µg 1 µl mg Direct-zol -96 MagBead RNA Kit R21, R211* R212, R213* R214, R215* 8 x 96 preps 1 µg 5 µl mg * Supplied with TRI Reagent. Compatible with samples stored in TRIzol, TRI-Reagent, RNAzol, QIAzol, and all other acid-guanidinium-phenol reagents. 3

6 Adjust to your surroundings. RNA from any Sample. Quick-RNA Kits (cells, tissues, biological fluids) Broad Range: Extract total RNA (including small/micro RNA) from any sample. DNA-Free: Genomic DNA removal column and DNase I included. NGS-Ready: RNA is ready for all downstream applications including Next-Gen Sequencing, RT-qPCR, hybridization, etc. 4

7 Universal Sample Compatibility [nt] Mammalian Cells Solid Tissue Whole Blood Bacteria, Yeast, Plant HeLa HCT116 WBC Liver Lung Spleen Brain Muscle Heart Human Porcine E. coli L. fermentum S. cerevisiae Nicotiana sp. The Quick-RNA Miniprep Plus Kit is universal and accommodates any sample input including cultured cells, any tissue, whole blood, tough-to-lyse samples, etc. DNA-Free RNA NGS-Ready RNA 4 3 Quick-RNA RT-PCR Quick-RNA Miniprep Plus Kit Kit 1 Kit 2 Kit 3 RFU 2 1 PCR Liver Lung Spleen Brain Muscle Heart Liver Lung Spleen Brain Muscle Heart Cycles RNA isolated with Quick-RNA is DNA-free compared to using. Total RNA was isolated from 1 6 human epithelial cells (with in-column DNase treatment for both kits, n=3). High-quality RNA was consistently isolated with the Quick-RNA Miniprep Plus Kit (left), but not with kits (right). Quality was assessed using the Agilent 22 TapeStation system; red = low quality. Product Cat. No. Size Binding Capacity Minimum Elution (Animal) Cells Tissue Sample Inputs Quick-RNA Microprep Kit R15 R151 1 µg 6 µl mg Low input; LCM Quick-RNA Miniprep Kit R154 R155 1 µg 5 µl mg Cells; Soft Tissue Quick-RNA Miniprep Plus Kit R157 R158 1 µg 5 µl mg Cells; Any Tissue; Whole Blood Quick-RNA Midiprep Kit R156 preps 1 mg 2 µl mg Cells; Soft Tissue Quick-RNA 96 Kit R152 R153 1 µg µl mg Cells; Soft Tissue 5

8 Quick-DNA/RNA (cells, tissues, biological fluids, LCM * ) Kits Highest Yields: Efficient isolation of genomic DNA and total RNA (including mirna) from any sample source. NGS-Ready: DNA/RNA is ready for all downstream applications including Next-Gen Sequencing, RT-qPCR, hybridization, etc. Single-Cell Detection: Ready for high-sensitivity assays down to a single-cell (6 µl elution volume). [bp] Mammalian Cells Solid Tissue Whole Blood Bacteria, Yeast, Plant Universal Sample Compatibility The Quick-DNA/RNA Plus technology is universal and accommodates any sample input including cultured cells, any tissue, blood, tough-to-lyse samples, etc. RNA DNA [nt] HeLa HCT116 WBC Liver Lung Spleen Brain Muscle Heart Human Porcine E. coli L. fermentum S. cerevisiae Nicotiana sp. Highest Yields Single-Cell Detection Yield (µg) DNA RNA Ct DNA RNA R² =.9859 R² = Cell Number Input DNA and total RNA recovery is higher using the Quick-DNA/RNA Microprep Plus Kit compared to a kit. Nucleic acids were extracted from 5K HeLa cells (n=2). HeLa cells were titrated down to a single cell, and DNA and total RNA were extracted using the Quick-DNA/RNA Microprep Plus Kit. Analysis by RT-qPCR shows high linear recovery of DNA & RNA down to the single-cell level (n=2). Product Cat. No. Size Binding Capacity Minimum Elution (Animal) Cells Tissue Format Quick-DNA/RNA Microprep Plus Kit D75* 1 µg 6 µl mg Column Quick-DNA/RNA Miniprep Kit D71 5 µg µl 5 x 1 6 mg Column Quick-DNA/RNA Miniprep Plus Kit D73 1 µg 5 µl mg Column Quick-DNA/RNA MagBead Kit R213 R x 96 preps 2 µg 5 µl mg Magbead * Compatible with Laser Capture Microdissection samples. 6

9 RNA Clean & Concentrator (any RNA, enzymatic reactions, TRIzol aqueous phase) Kits NGS-Ready: RNA is ready for all downstream applications including Next-Gen Sequencing, RT-qPCR, hybridization, etc. Ultra-Pure: Eliminate contaminants and inhibitors in 5 minutes. Maximum Recovery: Recover >9% and elute in as little as 6 μl. 67% used for library prep Widely Used for NGS 52% used in Illumina protocols Based on cited journal articles, 67% of all RNA Clean & Concentrator (RCC ) kits are used for library prep, and 52% of the same set of citations are used for Illumina protocols and workflows. RCC Usage Ultra-Pure High Recovery Rate RCC Purity (A26/23) RNA Recovery (%) RCC.2 1 RNA Input (µg) 5 1 RNA was cleaned-up using the RCC kit and a kit (n=4). RNA purity (measured by A26/23) was greater than 1.8 for the RCC kit but not for the kit. Increasing amounts of RNA was cleaned up using the RCC kit and a kit (n=2). RCC provides higher yields and more consistent recovery when compared to the kit. Product Cat. No. Size Binding Capacity Minimum Elution RNA Clean & Concentrator -5 Kit R113*, R115 R114*, R116 1 µg 6 µl RNA Clean & Concentrator - Kit R117 R118 1 preps 5 µg µl RNA Clean & Concentrator -1 Kit R119 preps µg 1 µl ZR-96 RNA Clean & Concentrator -5 Kit R18 µg 1 µl RNA Clean & Concentrator MagBead Kit R181 R182 1 ml 5 ml 1 µg 1 µl * Supplied with DNase I. 7

10 Quick-DNA/RNA (plasma, serum) Viral Kits Sample Input: Compatible with plasma/serum, cell culture media, biological fluids, swabs, feces. Streamlined Workflow: Sample inactivation and easy one-step lysis enables fast processing. High-Sensitivity: Optimized for low viral copy detection for Next-Gen Sequencing and RT-qPCR. 4. High-Sensitivity Viral Detection from Plasma 35. The Quick-RNA Viral Kits ensure high-sensitivity viral detection compared to the kit. Viral RNA was isolated from plasma samples. Data shows the mean (+/- SD) of triplicate RT-qPCR measurements. Cq Viral Copies (HIV) Quick-DNA/RNA (insect vectors, tissues) Pathogen Kits Sample Input: Compatible with vectors (mosquitoes, fleas, ticks) and tissues (mammals, birds). Streamlined Workflow: Sample inactivation and easy one-step lysis enables fast processing. Reliable Detection: Optimized for pathogen (virus, bacteria, protozoa) detection for Next-Gen Sequencing and RT-qPCR. 4 WNV spiked-in mosquitoes WNV (no mosquitoes) Inhibitor-free Detection in Mosquitoes 35 3 Hard-to-lyse and inhibitor-rich mosquito vectors were homogenized using ZR BashingBeads and purified using the Quick-DNA/RNA Pathogen Kit. Ultra-pure, inhibitor-free West Nile Virus nucleic acids (spike-in) were detected by RT-qPCR down to 4 viral copies. Cq x1e7 4x1e6 4x1e5 4x1e4 4, 4 4 Viral Copies (West Nile Virus) Product Cat. No. Size Binding Capacity Minimum Elution Input Amount Format Quick-RNA Viral Kit (for plasma/serum, biological fluids) R134 R14 1 µg 1 µg 6 µl 1 µl 2 µl 2 µl Column Plate Quick-DNA/RNA Viral Kit (for plasma/serum, biological fluids) D72 D722 R214 preps 96 preps 5 µg 1 µg 1 µg 35 µl 1 µl 5 µl 2 µl 2 µl 4 µl Column Plate Magbead Quick-DNA/RNA Pathogen Kits (for insect vectors and tissues) R142 R preps 5 µg 1 µg µl 5 µl 1 mg 1 mg Column Magbead Additional sizes available (see Page 13) 8

11 Environmental RNA Kits (bacteria, fungi, plant, soil) High-Quality: Extract high-quality total RNA (including small/micro RNA) from any environmental sample. Homogenization: Ultra-high density BashingBead technology for complete disruption and sample lysis. Inhibitor-Free: RNA is ready for all downstream applications including Next-Gen Sequencing, RT-PCR, microarray, etc. [nt] High-Quality RNA High-quality total RNA was isolated from different microbial species including Gram-negative bacteria (E. coli, P. aeruginosa, S. enterica), Gram-positive bacteria (E. faecalis, L. monocytogenes, L. fermentum), and yeast (S. cerevisiae) using the Quick-RNA Fungal/Bacterial Miniprep Kit. RNA was visualized using the Agilent 22 TapeStation system. 5 2 Complete Homogenization E. coli P. aeruginosa S. enterica E. faecalis L. monocytogenes L. fermentum S. cerevisiae Inhibitor-Free Zymo-Spin III-HRC Treated Samples ((( ))) Flourescence Untreated Samples X X Cycles State-of-the-art BashingBeads are ideal for disrupting tough-to-lyse organisms when paired with bead mills or high speed cell disrupters. Quick-RNA Environmental Kits removed PCR inhibitors using the Zymo-Spin III-HRC Filter. RNA was isolated from a Nicotiana sp. leaf, spiked with humic acid, with and without the Zymo-Spin III-HRC Filter. RT-qPCR detected early amplification for the filter treated samples compared to the untreated samples (c p = 3 vs. 31). Product Cat. No. Size Binding Capacity Minimum Elution Input Amount Quick-RNA Fungal/Bacterial Miniprep Kit (for Gram-negative/positive bacteria, yeast, fungi) R214 5 µg µl 5-1 mg wet weight Quick-RNA Plant Miniprep Kit (for leaves, stems, buds, flowers, fruit, seeds) Quick-RNA Tissue/Insect Microprep Kit (for insect and arthropod specimens, tissues) Quick-RNA Fecal/Soil Microbe Microprep Kit (for feces, soil, sludge, sediments) R224 5 µg µl 15 mg R23 1 µg 6 µl 1 mg R24 1 µg 6 µl mg 9

12 Quick-RNA Blood Kits (fresh or anticoagulated blood, blood cells) Superior Yields: Recover total RNA (including small/micro RNA) without sample loss. Protection: Worry-free blood sample storage at ambient temperatures for up to 3 days. High-Quality: RNA is ready for all downstream applications including Next-Gen Sequencing, RT-PCR, etc. [nt] High-Quality RNA High-quality RNA was extracted from human whole blood using the Quick-RNA Whole Blood Kit. Blood was stored in DNA/RNA Shield at ambient temperatures for two days prior to extraction (n=4). RNA was visualized using the Agilent 22 Tapestation system Superior Yields Protection RNA Degraded 5 35 Yield (µg) 4 3 Ct 3 RNA Stabilized Day Day 5 Day 7.5 ml 1 ml Amount of RNA extracted from 1 ml of human whole blood was significantly higher using the Quick-RNA Whole Blood Kit vs the kit (n=3). RT-qPCR shows the workflow stabilizes RNA, while the workflow leads to degradation. Whole blood was stored up to 7 days at ambient temperatures and extracted at the indicated time points using the Zymo Research or preservatives and workflows. Product Cat. No. Size Binding Capacity Minimum Elution Input Amount Quick-RNA Whole Blood Kit (compatible with EDTA, citrate, and heparin blood) Quick-DNA/RNA Blood Tube Kit (for use with the DNA/RNA Shield - Blood Collection Tube) DNA/RNA Shield Blood Collection Tube (collect and stabilize 3 ml blood in vacuum tubes): isolate up to 3 ug DNA and 3 ug RNA R121 1 µg 6 µl 1 ml R µg 1 µl 3 ml blood R115 5 tubes 1

13 Quick-cfRNA (serum, plasma, saliva, cerebrospinal fluid, amniotic fluid) Serum & Plasma Kits Quick & Easy: Simple spin-column based isolation. No phenol/chloroform or precipitation. Highest Yields: Efficient isolation of total cell-free RNA. Recover up to 515 times more microrna. Ultra-Pure: Ready for RT-qPCR, Next-Gen Sequencing, ncounter, etc. Highest Recovery of Cell-Free mirna 1.E+1 Copy number, hsa-mir-16-5p 8.E+9 6.E+9 4.E+9 2.E+9.E+9 Quick-cfDNA/RNA.5 ml Quick-cfDNA/RNA 1. ml Quick-cfDNA/RNA 3. ml (max) mir Adv Kit.2 ml mir Adv Kit.6 ml (max) QA CNA Kit 1. ml QA CNA Kit 3. ml (max) Cell-free RNA recovery scales proportionally with sample input using the Quick-cfDNA/cfRNA Serum & Plasma Kit. Cell-free RNA yields from the same plasma donor (61y-F) show linear and efficient recovery of plasma microrna (hsa-mir-16-5p) when analyzed by RT-qPCR. Ultra-Pure & Ready for RT-qPCR Compatible with Various Biological Fluids RFU [bp] (1 ml) Supplier M (.9 ml) Cycles ml 3 ml 1 ml 3 ml 1 ml 3 ml AF CSF Media The Quick-cfRNA Serum & Plasma Kit results in high purity and recovery of plasma microrna (hsa-mir-16-5p) based on RT-qPCR analysis. Amniotic fluid (AF), cerebrospinal fluid (CSF), or spent HeLa cell culture media (Media) were used to isolate cell-free nucleic acids using Quick-cfDNA/cfRNA Serum & Plasma Kit. Endogenous cell-free DNA from each sample type was visualized using the Agilent 22 Tapestation system. Product Cat. No. Size Binding Capacity Minimum Elution Input Amount Quick-cfRNA Serum & Plasma Kit R159 1 µg 6 µl 1 ml Quick-cfDNA/cfRNA Serum & Plasma Kit R172 1 µg 6 µl 3 ml 11

14 Quick-DNA/RNA (fixed & embedded tissues) FFPE Kits Easy Processing: Includes Deparaffinization Solution for simple paraffin removal. No xylene necessary. Improved Recovery: Optimized Proteinase K digestion ensures maximum recovery. High-Quality: Non-crosslinked DNA and DNA-free RNA are ready for all downstream applications including PCR, Next-Gen Sequencing, etc. Improved Recovery RFU DNA Cycles RFU RNA Avg Ct = Cycles DNA & RNA isolated using the Quick-DNA/RNA FFPE Kit are high quality and consistently outperforms RNA isolated using a kit (Avg Ct = 3.6) as depicted by the RT-PCR amplification curves (n=4). High-Quality FFPE DNA High-Quality FFPE RNA 1 bp DNA >5 bp 1 kb DNA >5 bp [nt] bp 2 Equivalent amounts of DNA resolved in a 1% agarose/tae/etbr gel show binding conditions may be adjusted with the Quick-DNA FFPE Kit to selectively isolate DNA >5 bp or >5 bp. 1 bp and 1 kb DNA ladder shown. RNA isolated with the Quick-RNA FFPE Kit is higher quality (left) compared to a kit (right). Quality assessed using the Agilent 22 TapeStation system. Product Cat. No. Size Binding Capacity Minimum Elution Input Amount Quick-DNA FFPE Kit D367 µg µl mg Quick-RNA FFPE Kit R18 5 µg µl mg Quick-DNA/RNA FFPE Kit R19 5 µg µl mg 12

15 RNA Isolation Products Product Cat. No. Size Product Cat. No. Size Direct-zol RNA Microprep Kit R26, R261* R262, R263* Quick-DNA/RNA Viral Kit D72 D721 preps 1 preps Direct-zol RNA Miniprep Kit R25, R251* R252, R253* Quick-DNA/RNA Viral 96 Kit D722 D723 Direct-zol RNA Miniprep Plus Kit Direct-zol -96 RNA Kit Direct-zol -96 MagBead RNA Kit R27, R271* R272, R273* R254, R255* R256, R257* R21, R211* R212, R213* R214, R215* 8 x 96 preps Quick-DNA/RNA Viral MagBead Kit Quick-DNA/RNA Pathogen Miniprep Kit Quick-DNA/RNA Pathogen MagBead Kit R214 R2141 R142 R143 R2145 R x 96 preps 1 x 96 preps Quick-RNA Fungal/Bacterial Microprep Kit R21 Quick-RNA Microprep Kit R15 R151 Quick-RNA Fungal/Bacterial Miniprep Kit R214 Quick-RNA Miniprep Kit R154 R155 Quick-RNA Plant Miniprep Kit R224 Quick-RNA Miniprep Plus Kit R157 R158 Quick-RNA Tissue/Insect Microprep Kit R23 Quick-RNA Midiprep Kit R156 preps Quick-RNA Fecal/Soil Microbe Microprep Kit R24 Quick-RNA 96 Kit R152 R153 Quick-RNA Whole Blood Kit R121 Quick-DNA/RNA Microprep Plus Kit D75 Quick-DNA/RNA Blood Tube Kit R1151 Quick-DNA/RNA Miniprep Kit D71 Quick-cfRNA Serum & Plasma Kit R159 Quick-DNA/RNA Miniprep Plus Kit D73 Quick-cfDNA/cfRNA Serum & Plasma Kit R172 Quick-DNA/RNA MagBead Kit R213 R x 96 preps Quick-RNA FFPE Kit R18 RNA Clean & Concentrator -5 Kit RNA Clean & Concentrator -5 Kit w/ DNase RNA Clean & Concentrator - Kit R115 R116 R113 R114 R117 R118 1 preps RNA Clean & Concentrator -1 Kit R119 preps ZR-96 RNA Clean & Concentrator -5 Kit R18 RNA Clean & Concentrator MagBead Kit ssdna/rna Clean & Concentrator Quick-RNA Viral Kit Quick-RNA Viral 96 Kit R181 R182 D71 R711 R134 R135 R14 R141 1 ml 5 ml 2 preps Quick-DNA/RNA FFPE Kit R19 ZR Urine RNA Isolation Kit R138 R139 2 preps YeaStar RNA Kit R12 4 preps ZymoBIOMICS RNA Miniprep Kit R21 ZymoBIOMICS DNA/RNA Miniprep Kit R22 ZymoBIOMICS DNA/RNA MagBead Kit R2135 R x 96 preps Pinpoint Slide RNA Isolation System I R13 Pinpoint Slide RNA Isolation System II R17 Zymoclean Gel RNA Recovery Kit R111 ZR small-rna PAGE Recovery Kit R17 2 preps * Supplied with TRI Reagent. 13

16 Cat. No. B13 Version 2 The BEAUTY of SCIENCE is to Make Things SIMPLE info@zymoresearch.com Toll Free: (888)