HuluFISH Kit Protocol – On Chip
INTRODUCTION
Document: HuluFISH Kit Protocol – On Chip
Version: 0.3
Release date: 2019/01/01
Associated product: HuluFISH Kit
HuluFISH is industry’s first multiplexing single molecule FISH (smFISH) probe developed by PixelBiotech GmbH. HuluFISH can be used to detect DNA/RNA expression in digital quantification and the sample can be in isolated DNA/RNA, fixed cell, fixed tissue sections or fixed whole mount embryo.
This manual is for detection of RNA/DNA on-chip by HuluFISH. The following protocols are included: HuluFISH staining.
HULUFISH STAINING
Step 1: Probe preparation
Resuspend HuluFISH probe in 33 ul DNase/RNase Free water (i.e. DEPC treated water or commercial water aliquots for molecular biology use).
TIPS: Facilitate the dissolution of HuluFISH probe in water by tapping the tube for several times. Alternatively, leave the tube on bench at room temperature for 20 min. The HuluFISH probe should be stored at -20 degree or lower. It is ok for repeated use by freezing-and-thawing the probe at room temperature.
Step 2: DNA/RNA on-chip capture
Take 1 ul of isolated RNA/DNA target in solution and hybridize with the glass slide immobilized with capture probe for the target. Hybridize in humidified chamber at 37 degree for 1 hour.
TIPS: Design a capture probe for the target. The capture probe should contain a 5’ end T10C10 tag for immobilization on regular glass slide, then followed by a sequence complementary to the 5’ end sequence of the target (20 bp should be enough). Immobilizing the capture probe on clean glass slide by uv crosslinking according to published reference (Biotechniques. 2008 Sep;45(3):261-71. doi: 10.2144/000112905).
Step 3: Washing the unbound target
Wash the glass slide with 2xSSC, 10% formamide, 0.1 % Tween-20 (WashT) for 2 times, each time 10 min at room temperature.
TIPS: WashT should be prepared with DEPC treated water to eliminate any RNase/DNase contamination.
Step 4: Staining with HuluFISH probe
Dilute 0.5 ul of HuluFISH probe in step 1 into 50 ul 1xHyb solution (2xSSC, 10% formamide, 10% dextran sulfate, 1mg/ml tRNA E. coli, 2mM RVC complex, 0.2mg/ml BSA). Take 10 ul HuluFISH working solution on the spot of captured target on glass slide. Hybridize in humidified chamber at 37 degree for 2 hours.
TIPS: In order to minimize solvent evaporation during hybridization, one can reduce the hybridization temperature to 30 degree, or cover the solution with one 13mm coverslip.
Step 5: Washing the unbound probe
Wash the glass slide with 2xSSC, 10% formamide, 0.1 % Tween-20 (WashT) for 4 times, each time 10 min at room temperature.
TIPS: See tips in step 3.
Step 6: Mounting
Remove the residual buffer on the spot of target. Pipette 10 ul Prolong Gold/Glass mounting solution on the spot and immediately cover with one 13mm coverslip (#1.5 or #1.0). Allow the sample to cure for 24-48 hours according to the instruction from Prolong Gold/Glass.
TIPS: Imaging the target on chip by epifluorescence or confocal microscope with appropriate laser. HuluFISH probe is labelled with the combination of Atto488, Atto565 and Atto647N. All fluorophores are barcoded as G (Atto488), Y (Atto565), and R (Atto647N). Check your probe barcoding scheme on the tube labe. For example, Gapdh-1G1Y1R is standing for mouse Gapdh HuluFISH probe with one Atto488, one Atto565 and one Atto647N. GAPDH-2G1R is standing for Human GAPDH gene with 2 Atto488 and one Atto647N.
Appendix
Appendix 1: Recommended Reagents from other vendors