DP419 RNAsimple Total RNA Kit. RNAprep pure Series. DP501 mircute mirna Isolation Kit. DP438 MagGene Viral DNA / RNA Kit. DP405 TRNzol Reagent

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1 Overview of TIANGEN Products DP419 RNAsimple Total RNA Kit DP430 RNAprep pure Kit(For Cell/Bacteria) DP315/DP315-R TIANamp Virus DNA/RNA Kit DP431 RNAprep pure Kit (For Tissue) Silica-membrane Technology RNAprep pure Series DP432 RNAprep pure Kit (For Plant) DP422 TIANamp DNA / RNA Isolation Kit DP433 RNAprep pure Kit (For Blood) DP423 TIANamp DNA/RNA/Protein Isolation Kit DP420 RNAprep pure Kit (For Micro Sample) Magnetic Particle Technology DP501 mircute mirna Isolation Kit DP438 MagGene Viral DNA / RNA Kit DP439 RNAprep pure FFPE Kit Phenol/Chloroform Extraction DP405 TRNzol Reagent DP421 TRNzol-A+ Reagent DP419 RNAsimple Total RNA Kit DP437 PNAplant plus Reagent DP501 mircute mirna Isolation Kit DP408 RNAstore Reagent Sample Stabilization DP440 RNALock Reagent DP409 RNAsafe (RNase Inhibitor) RNA Protection DP418 RNasin (RNase Inhibitor) RNA Cleanup DP412 RNAclean Kit 30

2 Overview of Selection guide for RNA purification products RNAprep pure Kit (For Tissue) RNAprep pure Kit (For Plant) RNAprep pure Kit (For Cell/Bacteria) RNAprep pure Kit (For Blood) RNAprep pure Kit (For Micro Sample) RNAprep pure FFPE Kit RNAsimple Total RNA Kit TIANamp Virus DNA / RNA Kit TIANamp Virus RNA Kit TIANamp DNA / RNA Isolation Kit TIANamp DNA / RNA / Protein Isolation Kit mircute mirna Isolation Kit TRNzol Reagent TRNzol-A+Reagent RNAplant plus Reagent MagGene Viral DNA/RNA Kit DP431 DP432 DP430 DP433 DP420 DP439 DP419 DP315 DP315-R DP422 DP423 DP501 DP405 DP421 DP437 DP438 Method Spin column Precipitation mag RNA purification Beads animal tissue Bacteria Plant Plant rich in secondary metabolites culture cell Yeast Blood micro Samples Virus Rna (serum, plasma, fluid) FFPE Samples Recommended kit compatible kit 31

3 Overview of RNA product feature companrison chart Cat.Num. Product Method Cell lysis Time Safety Convenience DP405 TRNzol Reagent Precipitation Guanidinium isothiocyanate / phenol 60 min DP421 TRNzol-A+ Reagent Precipitation Guanidinium isothiocyanate / phenol 60 min DP437 RNAplant plus Reagent Precipitation Surface-active / β-mercaptoethanol 90 min DP438 MagGene Viral DNA/RNA Kit Mag Beads Guanidine / SDS 45 min DP419 RNAsimple Total RNA Kit Spin Column Guanidinium isothiocyanate / phenol 60 min DP315 TIANamp Virus DNA / RNA Kit Spin Column Guanidine / SDS 60 min DP315-R TIANamp Virus RNA Kit Spin Column Guanidine 60 min DP422 TIANprep DNA/RNA Isolation Kit Spin Column Guanidine / β-mercaptoethanol min DP423 TIANprep DNA/RNA/Protein Isolation Kit Spin Column Guanidine / β-mercaptoethanol 60 min DP430 RNAprep pure Kit (For Cell/Bacteria) Spin Column Guanidine / β-mercaptoethanol min DP431 RNAprep pure Kit (For Tissue) Spin Column Guanidine / β-mercaptoethanol min DP432 RNAprep pure Kit (For Plant) Spin Column Guanidine / β-mercaptoethanol min DP433 RNAprep pure Kit (For Blood) Spin Column Guanidine / β-mercaptoethanol min DP420 RNAprep pure Kit (For Micro Sample) Spin Column Guanidine / β-mercaptoethanol min DP439 RNAprep pure FFPE Kit Spin Column Guanidine 3 hours DP501 mircute mirna Isolation Kit Spin Column Guanidinium isothiocyanate / phenol 60 min Cat.Num. Product Usage DP408 RNAstore Reagent RNA Stabilization for tissue samples storage DP409 RNAsafe ( RNase Inhibitor ) RNA Protection for RNA store DP418 RNasin ( RNase Inhibitor ) RNA Protection for RT reaction DP412 RNAclean Kit RNA Cleanup DP440 RNALock RNA Protection for blood samples storage 32

4 Overview of Introduction to Technology Principles and Methods RNA can be divided into messenger RNA, transfer RNA and ribosomal RNA. Purification of RNA is a series of processes of cell lysis, releasing RNA, eliminating contaminates, including protein, DNA, and other impurities, and obtaining high-purity RNA. Purification Process Sample preparation Purify RNA from samples of different sources (such as bacteria, yeast, blood, animal tissues, plant tissues and cultured cells), or of the same source in different forms (such as fresh blood, frozen blood, blood clots and dried blood). Reagent extraction Cell lysis Kit extraction Requirements for samples Fresh samples or fresh samples frozen at -80 immediately after collection are the most suitable sources for RNA purification. Repeated freeze-thaw circles should be avoided, which led to RNA degradation and low yields. Sample pre-treatment methods Plant sample Liquid nitrogen grinding Animal sample Tissue homogenation, liquid nitrogen grinding Extraction Add spin column Bacteria sample Lysozyme treatment Yeast sample Liquid nitrogen grinding, glass beads or lysozyme treatment Precipitation Remove protein Cell lysis Guanidine isothiocyanate / Phenol method Phenol / chloroform extraction is a liquid / liquid extraction Wash technique in biochemistry. It is widely used in molecular biology for RNA isolating. This method relies on phase separation by Wash centrifugation of a mix of the aqueous sample and a solution containing water-saturated phenol, chloroform and a chaotropic denaturing solution (Guanidine isothiocyanate) resulting in an upper aqueous phase and a lower organic phase (mainly Dissolved RNA chloroform). RNA are in the aqueous phase, while proteins remain in organic phase. In the next step, RNA is recovered from the aqueous phase by precipitation with isopropanol or Elution ethanol. TRNzol (DP405), TRNzol-A+ (DP421), and RNAsimple (DP419) are RNA purification kits developed based on guanidine isothiocyanate / phenol method. TRNzol is widely used for animal Purified RNA tissues, microbial tissues, cultured cells and plant that are not abundant in secondary metabolites. Purified RNA 33

5 Overview of Guanidine salt / β-mercaptoethanol method Suitable for purifying RNA from animal tissues and plants not abundant of secondary metabolites.β-mercaptoethanol is used as a denaturing reagent to inhibit RNase activity and keep RNA intact from degradation. TIANGEN RNAprep pure series purification kits are based on this method for RNA isolation from various samples. RNA abundance. It is recommended to increase starting tissue amount, and take complete grinding in liquid nitrogen. Tissues rich in protein or fat Fat content is high in brain or plant. Phenol / chloroform extraction should be done repeatedly for purifying the supernatant. Other methods Some plant sampels are rich in polysaccharide and polyphenol such as fruits and tomato leaves; some have high degree of lignification such as roots organization. RNAplant plus (DP437) are specially developed by TIANGEN for these samples. It can easily extract high-quality intact total RNA from plants rich in secondary metabolites. Requirements for purity No inhibitory organic solutions; low concentration of salts in samples No contamination of biological macromolecular such as protein, polysaccharide, and lipids No DNA molecule contamination Purification method and precipitation Method one: organic solvent extraction Phenol / chloroform extraction Phenol / chloroform extraction is a classical method to efficiently eliminate proteins from nucleic acid samples. During extraction, RNA migrates to the aqueous phase while DNA and proteins remain in the interphase or organic phase. TIANGEN s TRNzol and RNAplant plus are based on this method. Method two: Silica-membrane based adsorption With the improvement of technology, adsorption material based products have been developed for easier purification of nucleic acid. Silicon substrate adsorption is one of the most widely used methods due to its fast and simple features and no need of toxic organic compounds such as phenol and chloroform. RNAsimple Total RNA, RNAprep pure series are based on silicamembrane adsorption technology for RNA purification. Using specific spin columns and unique buffer system, protein and organic impurities are removed and high-quality RNA can be obtained. for Special Tissues Fibrous tissues Efficient cell lysis is vital for purifying RNA from heart and skeletal muscle fibrous tissues. These samples have low cell density and Nucleic acid / RNase rich tissues RNase content is rich in spleen, and thymus tissues, liquid nitrogen grinding and quick homogenate can effectively inactivate RNase. Repeated phenol / chloroform extraction can remove residual DNA. If white precipitate forms immediately after adding ethanol, that may indicate DNA contamination. This can be solved by using acid phenol / chloroform extraction or DNase Ⅰdigestion to eliminate DNA contamination. Plant tissues and fungal tissues RNA purification from plant tissues and fungal tissues are much more complicated than animal tissues. Sample grinding in liquid nitrogen is often necessary to prevent RNA from degradation. If RNA degradation cannot be solved by using this method, it is most likely caused by the impurities within the samples. Many plants and fungi are rich in polysaccharide and polyphenol which are difficult to be removed and may precipitate or bind with RNA. Extra steps or special reagents are needed (such as TIANGEN DP437 RNAplant plus) to remove polysaccharide and polyphenol contamination. RNA Identification and Evaluation Extracted RNA must take appropriate quality testing after purification to make sure it is suitable for downstream applications. RNA quality standards differ for various experiments. Intact RNA with no inhibitory impurities is required for cdna library construction. Intactness of RNA is vital for Northern blot, but inhibitory impurities are more tolerated. RT-PCR requires relatively low intactness level of RNA, but it is sensitive for inhibitory impurities. Different purification methods of RNA should be adapted for various downstream applications. RNA yield testing ultraviolet spectrophotometry RNA abundance is varied in different tissues. RNA concentration can be estimated using ultraviolet spectrophotometry which detects a linear change in absorbance with concentration. RNA has its absorption maximum at 260 nm and the ratio of the absorbance at 260 and 280 nm is used to assess the RNA purity. OD value at 260 nm is used to determine the RNA concentration in a solution. An OD 260 reading of 1.0 is equivalent to about 40 µg/ ml of RNA. The ratio of OD 260/280 of a pure RNA sample without protein contamination is between 1.8 and 2. Concentration of RNA (µg/ml) = OD 260 X 40 µg/ml X dilution ratio 34

6 Overview of Total RNA (µg) extracted from 1 mg sample High yields ( 4-2 µg ) Moderate yields ( µg ) Low yields ( µg ) Liver Brain Bladder Spleen embryo Bone Heart Kidney adipose Lung ovarian thymus Starting sample amount for RNA extraction Starting samples Sample amount Animal or plant tissues mg Mammal cells cells or 10 cm 2 culture plate G - bacteria cells or 1.8 ml culture (OD 600 = 2-3) G + bacteria cells or 1.8 ml culture (OD 600 = 1.8-2) Yeast cells cells or 1.5 ml culture (OD 600 = 1.8-2) RNA purity testing ultraviolet spectrophotometry Sample types rrna types Sizes ( Kb ) Ratio of OD 260 /OD 280 between 1.9 and 2.1, indicates high-purity of 18 S 1.9 Human the RNA; 28 S 5.0 Ratio of OD 260 /OD 280 less than 1.8, indicates protein impurity; 18 S 1.9 Ratio of OD 260 /OD 280 larger than 2.2, indicates RNA degradation; Mouse 28 S 4.7 Ratio of OD 260 /OD 280 less than 2.0, indicates residual guanidine isothiocyanate and β-mercaptoethanol. 18 S 2.0 Drosophila Attention: OD 260 /OD 280 values increase when buffer TE is used for 28 S 4.1 dissolution or elution of RNA. 16 S 1.5 RNA Integrity Identification Agarose gel electrophoresis Tobacco leaves 18 S S 2.9 Denaturing gel electrophoresis 25 S 3.7 We can identify RNA integrity by using denaturing gel 18 S 2.0 electrophoresis; but usually conventional electrophoresis testing Yeast 26 S 3.8 is also capable of doing the identification. rrna account for about 80%-85% of total RNA. 28S (23S) and 16 S 1.5 E.coli 18S (16S) rrna can be seen in agarose gel. 28S rrna is about 23 S 2.9 double amount of 18S rrna, which indicates the integrity of RNA. 18 S 1.8 Xenopus laevis rrna sizes from different soures are in the following table. 28 S 4.0 Conventional electrophoresis As denaturing gel electrophoresis process is much more complicated in conventional electrophoresis, therefore RNA integrity testing can be done in conventional electrophoresis, total RNA bands are the same for conventional electrophoresis and denaturing gel electrophoresis. RNA Protection RNA purification is much more complicated compared to DNA purification. It comes from the fact that RNA is very easily degradable because of the following Internal and External factors. 35

7 Overview of Internal factor: residues of nucleotide in RNA molecular have two hydroxyl groups, located at 2 and 3 positions respectively. The presence of these two hydroxyl groups makes RNA much more chemically reactive than DNA. External factor: RNase is widely existed in the surrounding environment. It cannot be easily inactivated by high temperature; therefore RNA becomes very prone to cleavage by contaminating RNases. RNA protection in purification process Sample homogenate Choose the right homogenate method to minimize the homogenate time and keep a low temperature. One of the most commonly used homogenate method is liquid nitrogen grinding. During grinding, don not let the liquid nitrogen volatilize completely and keep the sample frozen always. If the sample begins to thaw, RNA will be degraded by endogenous RNase. RNA protection prior to extraction Sample collection and storage Fresh sample is more appropriate for RNA purification. We should first select the fresh sample and take quick liquid nitrogen grinding and homogenate treatment to ensure the integrity of RNA in sample. If immediately purification of RNA is not possible after sample collection, proper storage method is necessary. Storing samples in liquid nitrogen is a commonly used method to prevent RNA degradation. Repeated freeze-thaw cycles should be avoided. RNase-free experimental condition RNase is abundant in natural environment and surrounding air. RNA purification experiment should be conducted in a special area which is free of RNase and prevented from crosscontamination with other experimental areas. Cell lysis Suitable and sufficient cell lysis buffer is important for cell lysis. The lysis buffers of TIANGEN s TRNzol, RNAsimple and RNAprep kits all have inhibitory effects on RNase. Sample amount should be in right proportion to lysis buffer. Too much sample amount led to inefficient cell disruption and compromised RNase inhibition, which cause low RNA yields and undermine the integrity of RNA. Storage condition After obtain the high-quality RNA, RNA protection is of great importance in the next stage. The most vital point is to prevent RNA from degradation. TIANGEN RNAsafe (DP409) provides a simple solution to this problem. RNAsafe is a unique chemical reagent which inhibits RNase and removes RNase contamination from solution to keep RNA intactness. It is compatible with various buffer solutions and further sterilization is no needed. RNase-free consumables and reagents Sterile, disposable plasticware is generally free of RNase contamination. Glassware should be treated by baking at 150 for >4 hours. In addition, treatment with 0.1% solution of diethylpyrocarbonate (DEPC), an inhibitor of RNase is sometimes used prior to autoclaving and baking glassware. Hands are a major source of RNase contamination. Use gloves at all stages of preparation. Segregate RNase-free glassware and reagents in the lab. DEPC treated H 2 O is also used for preparing reagents. Add 1ml DEPC per liter of water, incubate for 12 hours, and then autoclave for min. Use this treated water for preparing all solutions. Downstream applications RNA purification is the base for downstream molecular experiments, such as RT-PCR, Northern blot, in vitro transcription and translation. In these downstream applications, continued RNA protection is needed, and RNasin (DP418) is one of the widely used RNase inhibitor in the following experiments. 36