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Cost Model: 100 μl Reaction

Per-section reagent cost of the PRISM workflow, computed from the actual per-100 μl volumes in RCA Protocol: Direct RNA (v6.0) and Sample Pre-treatment: Fresh Frozen (v5.3), priced against vendor list prices. Companion to the Bill of Materials, which says what to order; this page says what it costs to run.

  • Scope: one FF section in one PDMS chamber -- 2.5 x 2.5 cm block, 5 mm punch = 98 μl, so the protocol tables are written for 100 μl
  • Route: direct-RNA library construction + PRISM single-round fluorescence readout
  • Price basis: China list prices as published by the vendor or its mainland distributor, August 2026. USD -> CNY at 6.74
  • Version: v1.2
  • Last modified: 2026-08-18

Re-quote before you budget

These are list prices captured on a single date. Institutional discounts, promotional pricing and distributor markups move them substantially -- Thermo's "loyalty" pricing alone is ~30% off several lines here. Treat the structure (which reagents dominate, and by how much) as the durable result and the absolute numbers as a snapshot.

How to use

Read Section 3 first. Three enzymes are 96% of the reaction cost, so nothing else is worth optimizing until those are addressed. Section 6 lists the levers with their measured savings.

1. Unit prices

Cost per μl is what matters, since every protocol table is written in μl.

Reagent Vendor / Cat # Pack Usable volume Price (CNY) CNY / μl
SplintR Ligase (25 U/μl) NEB M0375S 1,250 U 50 μl 1,2151 24.30
SplintR Ligase, bulk NEB M0375L 6,250 U 250 μl 5,2951 21.18
phi29 DNA Polymerase (10 U/μl) Thermo EP0091 250 U 25 μl 9262 37.04
RiboLock RNase Inhibitor (40 U/μl) Thermo EO0381 2,500 U 62.5 μl 3993 6.38
Yeast tRNA (10 μg/μl) Invitrogen AM7119 0.5 ml 500 μl 1,4554 2.91
Aminoallyl-dUTP (50 mM) Thermo R1101 50 μl 50 μl 1,7075 34.14
Ampligase 10X Reaction Buffer Biosearch A1905B 5 ml 5,000 μl ~1,300 (est.)7 0.26
Recombinant Albumin (20 μg/μl) NEB B9200S 12 mg 600 μl ~550 (est.)8 0.92
dNTP Solution Mix (10 mM each) NEB N0447S 8 μmol ea. 800 μl ~665 (est.)8 0.83
Formamide, deionized Sigma F9037 100 ml 100,000 μl 4029 0.0040
DAPI Thermo D1306 10 mg -- 2,0836 see DAPI Stock
SplintR Buffer 10X NEB, with M0375 -- -- 0 0
phi29 Buffer 10X Thermo B62, with EP0091 -- -- 0 0
KCl, glycerol, SSC, PBS-T, nuclease-free water bulk -- -- negligible <0.001

(est.) rows

Three items have no published mainland price. They are converted from the NEB/Biosearch US list price using the NEB China multiplier observed on SplintR (USD 95 -> CNY 1,215, i.e. ~12.8 CNY per catalog dollar -- roughly 1.9x the US price once converted at 6.74). All three together are under 2% of the reaction cost, so the estimate does not move the conclusion. Replace with a real quote when raising a PO.

2. Per-step cost of each 100 μl mix

Volumes are taken verbatim from the RCA v6.0 tables. Rows that cost effectively nothing (water, KCl, glycerol, bundled buffers) are omitted but still occupy their volume in the mix.

Step 1 -- Hybridization (excluding padlock probes)

Reagent Per 100 μl CNY / μl Cost (CNY)
Ampligase Buffer 10X 10 μl 0.26 2.60
Formamide 100% 20 μl 0.0040 0.08
Recombinant Albumin 1 μl 0.92 0.92
tRNA 2 μl 2.91 5.82
RiboLock 2.5 μl 6.38 15.96
Padlock probes (variable) -- see Section 5
Subtotal 25.38

Step 2 -- Ligation

Reagent Per 100 μl CNY / μl Cost (CNY)
SplintR Buffer 10X 10 μl 0 (bundled) 0
Recombinant Albumin 1 μl 0.92 0.92
SplintR Ligase 10 μl 24.30 243.00
RiboLock 2.5 μl 6.38 15.96
Subtotal 259.88

Step 3 -- Rolling Circle Amplification

Reagent Per 100 μl CNY / μl Cost (CNY)
phi29 Buffer 10X 10 μl 0 (bundled) 0
dNTPs 10 mM 2.5 μl 0.83 2.08
Aminoallyl-dUTP 2.5 μl of the 2 mM working stock = 0.1 μl of 50 mM 34.14 3.41
Recombinant Albumin 1 μl 0.92 0.92
RCA primer 10 μM 1 μl -- 0.06
phi29 Polymerase 2.5 μl 37.04 92.60
Subtotal 99.07

Three mixes = CNY 384.3 per section.

3. Where the money goes

Reagent Per section Share of the three mixes
SplintR Ligase (10 μl, 250 U) 243.00 63.2%
phi29 Polymerase (2.5 μl, 25 U) 92.60 24.1%
RiboLock (2.5 μl x 2 steps) 31.92 8.3%
Yeast tRNA (2 μl x 1 step) 5.82 1.5%
Ampligase Buffer (10 μl x 1 step) 2.60 0.7%
Recombinant Albumin (1 μl x 3 steps) 2.76 0.7%
Aminoallyl-dUTP 3.41 0.8%
dNTPs 2.08 0.5%
Everything else ~0.14 <0.1%

Three enzymes are 96% of the reaction cost. Every remaining reagent in the workflow combined -- buffers, carriers, nucleotides, formamide, all washes, the slide and the PDMS chamber -- comes to less than the SplintR in a single ligation.

The ligation step is worth a second look

10 μl of SplintR in 100 μl is 2.5 U/μl, about twice NEB's usual working range for a nick-ligation reaction. That one line item is 60% of the per-section cost. Whether it can be lowered on-slide is an empirical question -- on-section ligation is not a tube reaction, and the protocol may be carrying this concentration for a reason -- but it is the single highest-value titration available. See Section 6.

4. Full cost per section

Block What it covers Cost (CNY) Share
A. Three 100 μl reaction mixes Hybridization, ligation, RCA 384.33 94.2%
B. Washes and fixation Stringent wash (1 ml of 10% formamide / 2X SSC), 65% formamide strip (1 ml), post-amplification 4% PFA, ~30 PBS-T changes 3.40 0.8%
C. Pre-treatment (FF) 4% PFA in a slide mailer (6 ml, amortized over 5 slides), pepsin, ethanol series 5.34 1.3%
D. Readout ancillaries DAPI 100 μl at 10 ng/ml, 25 μl of 10% glycerol mountant 0.05 <0.1%
E. Consumables PDMS chamber (2.63), Superfrost Plus slide, coverslip, tips/tubes 14.63 3.6%
Total, excluding probes 407.75 (~USD 61)

DAPI is free, effectively

At the corrected 10 ng/ml working concentration (see DAPI Stock), 100 μl contains 1 ng of dye. One 10 mg vial of D1306 covers millions of sections. The superseded 5 μg/ml concentration would still have been under CNY 0.11 per section -- DAPI was never a cost question, only a crosstalk one.

5. Probes (amortized)

Probes are a capital cost, not a per-run one: you synthesize once and draw pmol-scale amounts per section. The per-section figure is therefore the one-off divided by how many sections a tube covers.

\[ \text{CNY per section} = \frac{N_{\text{plex}} \times \text{price per oligo}}{\text{delivered pmol} \;/\; (\,C_{\text{final}} \times 100\ \mu\text{l}\,)} \]

Worked for a 46-plex panel:

Per probe, per section Sections per tube One-off (CNY) Per section (CNY)
Padlock probes (46, ~90 nt, PAGE) at 10 nM each11 1 pmol ~5,000 ~18,400 ~3.7
PRISM detection probes (46 labelled + 46 plain) at _TODO_ -- -- -- _TODO_

The PRISM detection-probe concentration is not documented

PRISM Imaging specifies neither a working concentration nor a reaction table for the fluorescent detection probes -- only that 100 μl is applied per section. Until that number is recorded, the readout half of the probe cost cannot be computed. Illustratively, at the HybISS bridge-probe default of 0.1 μM each12 a 46-plex would consume 10 pmol per probe per section, giving ~1,000 sections from a 10 nmol tube and roughly CNY 69 per section against an assumed one-off of ~69,000 (CNY 1,200 per labelled oligo, 300 per plain). That is an assumption, not our value.

The oligo unit prices above (CNY 400 per padlock, 1,200/300 per detection probe) are industry-typical figures, not quotes. Substitute the real ones -- at these volumes the per-section amortization is small either way, but the one-off is five figures and worth quoting properly.

6. Reducing the cost

Each lever is independent; savings are against the CNY 384.3 reaction cost.

Lever Saves (CNY) Notes
Punch 4 mm instead of 5 mm (98 -> 63 μl) 138.4 Scales everything by (⅘)² = 0.64. Costs nothing but working area -- check it still covers the section
SplintR 10 -> 5 μl (2.5 -> 1.25 U/μl) 121.5 Highest-value titration. Validate on a control section before adopting
phi29 from a domestic supplier 74.6 Beyotime D7053S, 250 U at ~180 vs 92610. Requires a side-by-side rolony yield/size comparison
RiboLock from a domestic supplier 28.9 Commodity enzyme; ~0.6 CNY/μl is typical
Thermo loyalty pricing on EP0091 / EO0381 / AM7119 39.1 Purchasing change only, no protocol risk
Buy SplintR as M0375L 31.2 24.30 -> 21.18 CNY/μl. Only 13% -- check it will be used before it ages

Combining the SplintR halving, bulk SKU, and both domestic enzyme swaps takes the reaction cost from 384 to ~144 CNY (-63%); adding the 4 mm punch brings it to ~92 CNY. None of these are adopted -- each needs bench validation first.

Assumptions and limitations

  • Quoted from a vendor or mainland distributor page: SplintR (both SKUs), phi29, RiboLock, tRNA, aminoallyl-dUTP, DAPI, formamide.
  • Estimated (US list x the observed NEB China multiplier): Ampligase buffer, recombinant albumin, dNTP mix. Together <2% of the total.
  • Assumed, and the weakest numbers here: all oligo unit prices, the PRISM detection-probe concentration, 16% PFA and general consumable pricing.
  • Bundled buffers (SplintR, phi29) are costed at zero because they ship with their enzyme and are consumed at the same rate. If either is bought standalone, add it.
  • Not costed: labour, instrument time, imaging, storage, freezer/electricity, probe design, and the sequencing-side reagents that only SPRINTseq needs.
  • Pipetting dead volume and mix-preparation excess are not modelled. Preparing a mix for one section at a time realistically adds 10--20%; batching several sections into one mix largely removes it.

References

Vendor pages the prices were taken from, all accessed 2026-08-15.

  1. NEB SplintR Ligase, M0375S / M0375L -- Gene-Mart (NEB mainland distributor). https://www.gene-mart.cn/Product.aspx?id=562683
  2. Thermo Scientific phi29 DNA Polymerase (10 U/μL), EP0091. https://www.thermofisher.cn/order/catalog/product/cn/zh/EP0091
  3. Thermo Scientific RiboLock RNase Inhibitor (40 U/μL), EO0381. https://www.thermofisher.cn/order/catalog/product/cn/zh/EO0381
  4. Invitrogen Yeast tRNA (10 mg/mL), AM7119. https://www.thermofisher.cn/order/catalog/product/cn/zh/AM7119
  5. Thermo Scientific Aminoallyl-dUTP Solution (50 mM), R1101. https://www.thermofisher.cn/order/catalog/product/cn/zh/R1101
  6. Thermo Scientific DAPI, D1306. https://www.thermofisher.cn/order/catalog/product/cn/zh/D1306
  7. New England Biolabs, 2025 TCEFS academic price list (B9200S, N0447S, M0375S/L US list prices). https://www.umassmed.edu/globalassets/enzyme-freezer/documents/neb-tcefs-2025-pricing-list-01-29-25.pdf
  8. LGC Biosearch Technologies, Ampligase 10X Reaction Buffer, A1905B. https://www.biosearchtech.com/molecular-biology-enzymes/dna-and-rna-modifying-enzymes/dna-and-rna-ligases/ampligase-10x-reaction-buffer/p/A1905B
  9. Sigma-Aldrich Formamide, deionized, F9037. https://www.sigmaaldrich.com/US/en/product/sigma/f9037
  10. Beyotime phi29 DNA Polymerase, D7053S -- domestic alternative cited in Section 6. https://www.beyotime.com/product/D7053S.htm
  11. Gyllborg D, Nilsson M. HybISS: Hybridization-based In Situ Sequencing. protocols.io (2020). DOI: 10.17504/protocols.io.xy4fpyw. -- source of the 0.1 μM readout-probe figure used illustratively in Section 5.

Changelog

  • v1.2 -- 2026-08-18 -- Stringent wash recomputed at 10% formamide for RCA Protocol v7.0. MINOR: same model, one reagent volume halved. The 1 ml wash buffer now takes 100 μl of formamide instead of 200, so block B (washes and fixation) falls 3.80 → 3.40 and the per-section total excluding probes 408.15 → 407.75. Worth recording only because it is the arithmetic behind a bench-visible change; at 0.1% of the total, cost was never a reason to pick either stringency.

  • v1.1 -- 2026-08-18 -- Recomputed for RCA Protocol v6.0, which deletes the blocking step. MINOR: same model, one fewer mix. The reaction cost drops from four mixes at CNY 410.31 to three at 384.33, and the per-section total excluding probes from 434.13 to 408.15. Per-reagent lines follow: RiboLock now appears in two mixes rather than three (47.88 → 31.92), tRNA and Ampligase buffer in one rather than two, albumin in three rather than four. The enzyme share rises to 96% because a non-enzyme step was removed — the absolute enzyme cost is unchanged. The "drop RiboLock from blocking only" lever is retired with the step it applied to, and the combined-lever figure moves from ~155 to ~144 CNY (~92 with the 4 mm punch).

  • v1.0 -- 2026-08-16 -- Initial cost model. Built from the RCA v5.4 100 μl tables and FF pre-treatment v5.3, priced at August 2026 China list prices. Establishes that SplintR, phi29 and RiboLock are 94% of the reaction cost. Leaves the PRISM detection-probe concentration as _TODO_ -- it is not recorded anywhere on the site, and the readout half of the probe cost cannot be computed without it.

  1. Gene-Mart (NEB mainland distributor), SplintR Ligase M0375S store price CNY 1,215 (list 1,429) and M0375L CNY 5,295. US list for comparison: USD 95 / 420 (NEB 2025 price list). ↩↩

  2. thermofisher.cn, phi29 DNA Polymerase EP0091, 250 U: CNY 926 regular, 648 loyalty price. ↩

  3. thermofisher.cn, RiboLock RNase Inhibitor EO0381, 2,500 U: CNY 399 regular, 279 loyalty price. ↩

  4. thermofisher.cn, Yeast tRNA AM7119, 0.5 ml: CNY 1,455 regular, 1,038 loyalty price. ↩

  5. thermofisher.cn, Aminoallyl-dUTP Solution (50 mM) R1101, 50 μl: CNY 1,707. ↩

  6. thermofisher.cn, DAPI D1306, 10 mg: CNY 2,083 regular, 1,500 loyalty price. ↩

  7. LGC Biosearch Technologies A1905B, Ampligase 10X Reaction Buffer, 5 ml. No mainland list price published; US list USD 122.50. ↩

  8. New England Biolabs 2025 TCEFS price list: B9200S (Recombinant Albumin, 12 mg) USD 43; N0447S (dNTP mix, 8 μmol each) USD 52. ↩↩

  9. Sigma-Aldrich F9037, deionized formamide, 100 ml: ~CNY 402 from mainland resellers; US list ~USD 78-90. ↩

  10. Beyotime D7053S, phi29 DNA Polymerase, 250 U: ~CNY 163-192. Not validated against EP0091 in this lab. ↩

  11. RCA Protocol: Direct RNA v5.4 -- 10 nM per probe is the preferred overnight hybridization condition; the same-day option is 50 nM, which raises padlock consumption 5x. ↩

  12. Gyllborg D, Nilsson M. HybISS: Hybridization-based In Situ Sequencing. protocols.io (2020). DOI: 10.17504/protocols.io.xy4fpyw; manuscript Nucleic Acids Research 48(19), e112 (2020). DOI: 10.1093/nar/gkaa792. ↩

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