NovaFISH Kit Protocol – On Chip

INTRODUCTION

Document: NovaFISH Kit Protocol – On Chip

Version: 0.4

Release date: 2024/03/05

Associated product: NovaFISH Kit

NovaFISH is the industry’s first multiplexing single molecule FISH (smFISH) probe developed by PixelBiosciences GmbH. NovaFISH 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 the detection of RNA/DNA on-chip by NovaFISH. The following protocols are included: NovaFISH staining.

NovaFISH STAINING

Step 1: Probe preparation
Reconstitute NovaFISH probe in 33 μL DNase/RNase Free water (i.e. DEPC treated water or commercial water aliquots for molecular biology use).

TIPS: Facilitate the dissolution of NovaFISH probe in water by tapping the tube several times. Alternatively, leave the tube on bench at room temperature for 20 min. The NovaFISH probe should be stored at -20 ?C or lower. Repeated freezing-thawing cycles of the probe do not affect the product.

Step 2: DNA/RNA on-chip capture
Take 1μLl of isolated RNA/DNA target in solution and hybridize with the glass slide immobilized with a capture probe for the target. Hybridize in a humidified chamber at 37 ?C for 1 hour.

TIPS: Design a capture probe for the target. The capture probe should contain a 5’ end T10C10 tag for immobilization on a 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, formamide 10%, Tween-20 0.1 % (WashT) twice for 10 min each at room temperature.

TIPS: WashT should be prepared with DEPC-treated water to eliminate any RNase/DNase contamination.

Step 4: Staining with NovaFISH probe
Dilute 0.5 μL of NovaFISH probe in step 1 into 50 μL 1xHyb solution (2xSSC, formamide 10%, dextran sulfate 10%, tRNA E. coli 1 mg/mL, RVC complex 2mM, BSA 0.2mg/mL). Take 10 μL NovaFISH working solution on the spot of the captured target on a glass slide. Hybridize the preparation in a humidified chamber at 37 ?C for 2 hours.

TIPS: To minimize solvent evaporation during hybridization, it is possible to reduce the hybridization temperature to 30 ?C, or cover the solution with one 13 mm coverslip. 

Step 5: Washing the unbound probe
Wash the glass slide with 2xSSC, formamide 10%,Tween-20  0.1 %  (WashT) 4 times, each time for 10 min at room temperature.

TIPS: See tips in step 3.

Step 6: Mounting
Remove the residual buffer on the spot of the target. Pipette 10 μL of mounting medium on the spot and immediately cover with a 13 mm coverslip (#1.5 or #1.0). Store the sample at 4?C until use.

TIPS: Imaging the target on chip by epifluorescence or confocal microscope with appropriate laser. NovaFISH 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 stands for mouse Gapdh NovaFISH probe with one Atto488, one Atto565 and one Atto647N. GAPDH-2G1R stands for Human GAPDH gene with 2 Atto488 and one Atto647N.
 

Appendix

Appendix 1: Recommended Reagents from other vendors

l  Prolong Gold, ThermoFisher Scientific, Catalog Number P10144 (link)

l  Prolong Glass, ThermoFisher Scientific, Catalog Number P36982 (link)

l  RVC complex, NEB, Catalog Number S1402S (link)

l  BSA, Ambion, Catalog Number AM2616 (link)

l  tRNA E. coli, Sigma, Catalog Number 000000010109541001 (link)