Journal: Current Protocols

Article Title: Enzymatic Synthesis of High‐Density RNA Microarrays

doi: 10.1002/cpz1.667

Figure Lengend Snippet: Reaction Mixture for Hybridization of the Primer

Article Snippet: Final washing buffer (FWB; 0.1× SSC) RNase‐free water (Carl Roth, art. no. T143.5) RNase AWAY surface decontaminant (ThermoFisher Scientific, cat. no. 7002) Acetylated BSA (10 mg/ml) (Promega, cat. no. R3961) Primer oligonucleotide (2′ O ‐methyl RNA with 5′ psoralen‐C2 modification: 5′‐Ps‐UAGGGACACGGCGAA, 1 μM in RNase‐free water) (Eurogentec) 2× MES hybridization buffer (composition see below) Triton X‐100 (Sigma Aldrich, cat. no. T8787) DTT (0.1 M) (e.g., ThermoFisher Scientific, cat. no. 707265ML) RNase inhibitor, human placenta (40 U/μl) (NEB, cat. no. M0307) NTP set (100 mM each) (ThermoFisher Scientific, cat. no. R0481) Skirted reagent tubes with screw cap, 50 ml (e.g., Greiner bio‐one, VWR 525‐0396) Microcentrifuge tubes Biopur Safe‐Lock 1.5 ml (VWR 211‐2161) Micropipettes Sterile filter tips (Starlab S1121‐3810, S1120‐1840, and S1126‐7810) Hybridization oven (e.g., Boekel Scientific “Little Shot” 230500) DNA template microarray Self‐adhesive hybridization chambers, whose dimensions are adapted to the layout of the microarray (e.g., Grace Bio‐Labs SecureSeal SA200, RD500958 for Agilent SurePrint 4 × 44 K DNA microarray) Tweezers Adhesive seal tabs (Grace Bio‐Labs ST200) Aluminum foil Timer Slide spinner (Labnet International C1303‐T) Fridge or cold room at ∼4°C High‐power Nichia NVSU333A 365 nm UV LED in a custom‐made setup for photocrosslinking, as previously described in detail (Schaudy, Hölz, Lietard, & Somoza, ) and shown in Figure SÜSS Model 1000 UV intensity meter with a 365‐nm probe (alternative: Ushio UIT 201 Digital UV intensity meter with Ushio UVD‐365PD 365‐nm probe) NOTE: We recommend storing the reaction buffers and the acetylated BSA in smaller aliquots to avoid repeated freeze‐thaw cycles and allow for faster thawing.

Techniques: Hybridization

Journal: Current Protocols

Article Title: Enzymatic Synthesis of High‐Density RNA Microarrays

doi: 10.1002/cpz1.667

Figure Lengend Snippet: Pre‐Incubation Mixture

Article Snippet: Final washing buffer (FWB; 0.1× SSC) RNase‐free water (Carl Roth, art. no. T143.5) RNase AWAY surface decontaminant (ThermoFisher Scientific, cat. no. 7002) Acetylated BSA (10 mg/ml) (Promega, cat. no. R3961) Primer oligonucleotide (2′ O ‐methyl RNA with 5′ psoralen‐C2 modification: 5′‐Ps‐UAGGGACACGGCGAA, 1 μM in RNase‐free water) (Eurogentec) 2× MES hybridization buffer (composition see below) Triton X‐100 (Sigma Aldrich, cat. no. T8787) DTT (0.1 M) (e.g., ThermoFisher Scientific, cat. no. 707265ML) RNase inhibitor, human placenta (40 U/μl) (NEB, cat. no. M0307) NTP set (100 mM each) (ThermoFisher Scientific, cat. no. R0481) Skirted reagent tubes with screw cap, 50 ml (e.g., Greiner bio‐one, VWR 525‐0396) Microcentrifuge tubes Biopur Safe‐Lock 1.5 ml (VWR 211‐2161) Micropipettes Sterile filter tips (Starlab S1121‐3810, S1120‐1840, and S1126‐7810) Hybridization oven (e.g., Boekel Scientific “Little Shot” 230500) DNA template microarray Self‐adhesive hybridization chambers, whose dimensions are adapted to the layout of the microarray (e.g., Grace Bio‐Labs SecureSeal SA200, RD500958 for Agilent SurePrint 4 × 44 K DNA microarray) Tweezers Adhesive seal tabs (Grace Bio‐Labs ST200) Aluminum foil Timer Slide spinner (Labnet International C1303‐T) Fridge or cold room at ∼4°C High‐power Nichia NVSU333A 365 nm UV LED in a custom‐made setup for photocrosslinking, as previously described in detail (Schaudy, Hölz, Lietard, & Somoza, ) and shown in Figure SÜSS Model 1000 UV intensity meter with a 365‐nm probe (alternative: Ushio UIT 201 Digital UV intensity meter with Ushio UVD‐365PD 365‐nm probe) NOTE: We recommend storing the reaction buffers and the acetylated BSA in smaller aliquots to avoid repeated freeze‐thaw cycles and allow for faster thawing.

Techniques: Hybridization

Journal: Current Protocols

Article Title: Enzymatic Synthesis of High‐Density RNA Microarrays

doi: 10.1002/cpz1.667

Figure Lengend Snippet: Solution for Hybridization With a Fluorescently Labeled Complement

Article Snippet: Final washing buffer (FWB; 0.1× SSC) RNase‐free water (Carl Roth, art. no. T143.5) RNase AWAY surface decontaminant (ThermoFisher Scientific, cat. no. 7002) Acetylated BSA (10 mg/ml) (Promega, cat. no. R3961) Primer oligonucleotide (2′ O ‐methyl RNA with 5′ psoralen‐C2 modification: 5′‐Ps‐UAGGGACACGGCGAA, 1 μM in RNase‐free water) (Eurogentec) 2× MES hybridization buffer (composition see below) Triton X‐100 (Sigma Aldrich, cat. no. T8787) DTT (0.1 M) (e.g., ThermoFisher Scientific, cat. no. 707265ML) RNase inhibitor, human placenta (40 U/μl) (NEB, cat. no. M0307) NTP set (100 mM each) (ThermoFisher Scientific, cat. no. R0481) Skirted reagent tubes with screw cap, 50 ml (e.g., Greiner bio‐one, VWR 525‐0396) Microcentrifuge tubes Biopur Safe‐Lock 1.5 ml (VWR 211‐2161) Micropipettes Sterile filter tips (Starlab S1121‐3810, S1120‐1840, and S1126‐7810) Hybridization oven (e.g., Boekel Scientific “Little Shot” 230500) DNA template microarray Self‐adhesive hybridization chambers, whose dimensions are adapted to the layout of the microarray (e.g., Grace Bio‐Labs SecureSeal SA200, RD500958 for Agilent SurePrint 4 × 44 K DNA microarray) Tweezers Adhesive seal tabs (Grace Bio‐Labs ST200) Aluminum foil Timer Slide spinner (Labnet International C1303‐T) Fridge or cold room at ∼4°C High‐power Nichia NVSU333A 365 nm UV LED in a custom‐made setup for photocrosslinking, as previously described in detail (Schaudy, Hölz, Lietard, & Somoza, ) and shown in Figure SÜSS Model 1000 UV intensity meter with a 365‐nm probe (alternative: Ushio UIT 201 Digital UV intensity meter with Ushio UVD‐365PD 365‐nm probe) NOTE: We recommend storing the reaction buffers and the acetylated BSA in smaller aliquots to avoid repeated freeze‐thaw cycles and allow for faster thawing.

Techniques: Hybridization, Labeling