community.elabeln.com/experiments.php?mode=edit&id=314

#314 Molecular Biology Running

qPCR, HER2 Expression in HeLa vs. SK-BR-3

Started on

Tags

qpcr HER2 breast-cancer IFN-γ molecular-bio

Custom ID

Objective: Quantify HER2 / ERBB2 mRNA expression in HeLa (low expressor) vs. SK-BR-3 (HER2-amplified) cell lines, with and without 24 h IFN-γ stimulation. GAPDH is used as the endogenous reference for ΔΔCt normalization.

Reagents: 2× SYBR Green Master Mix (Thermo, A25742), HER2 fwd/rev primers (10 µM, IDT lot #20240118), GAPDH fwd/rev primers (10 µM, IDT lot #20240118), cDNA template (50 ng/µL, RT’d from 1 µg total RNA, see RT prep · EXP-2026-0831), nuclease-free water.

Reaction volume: 20 µL per well. Plate: 96-well, technical triplicates, 24 wells total. Instrument: QuantStudio™ 5 (Lab B-204).

Expected outcome: SK-BR-3 should show ~150-200× higher HER2 expression vs. HeLa baseline. IFN-γ treatment is expected to modestly upregulate HER2 (~3-5×) in both lines.

Last saved: 34 seconds ago

12 fields loaded from qPCR Template v3.2

Sample & Target

Cell line

select

HeLa, SK-BR-3

Treatment

text

IFN-γ 10 ng/mL, 24 h

Target gene

text

HER2 (ERBB2)

Reference gene

text

GAPDH

Reaction Conditions

Master mix

select

2× SYBR Green (Thermo A25742)

Reaction volume

number

20µL

Primer concentration

number

0.4µM

cDNA per well

number

5ng

Cycling Program

Initial denaturation

text

95 °C × 10:00

Cycles

number

40

Denature → Anneal

text

95 °C × 0:15 → 60 °C × 1:00

Melt curve

check

65 → 95 °C, +0.5 °C/step

plate_layout.pdf

142 KB

quantstudio_results.csv

512 KB

amp_curves_export.xlsx

1.2 MB

01

Thaw reagents on ice (SYBR mix, primers, cDNA)

Recorded at 08:14 by SC

0:10

02

Prepare master mix (24 wells × 1.1 excess)

0:15

03

Aliquot 18 µL master mix into 96-well plate

0:08

04

Add 2 µL cDNA per well (technical triplicates)

0:12

05

Seal plate with optical film, vortex, brief spin (1k × 30s)

0:04

06

Load plate into QuantStudio 5 (Lab B-204)

0:02

07

Run cycling program (validated: 40 cycles + melt)

Auto-pushed to instrument · run ID 2026-0310-08:22

1:35

08

Export raw .eds and CSV to ELabELN

0:02

09

Run ΔΔCt analysis (auto-linked GAPDH reference)

0:01

10

Apply e-signature for QA sign-off

Awaiting Dr. Chen

0:03

	A	B	C	D	E	F
1	Sample	Replicate	Target	Cq	ΔΔCt	Fold Change
2	HeLa	1	HER2	28.42	0.00	1.0
3	HeLa	2	HER2	28.51	0.06	0.96
4	HeLa	3	HER2	28.39	-0.02	1.02
5	SK-BR-3	1	HER2	20.81	-7.65	201.3
6	SK-BR-3	2	HER2	20.93	-7.53	184.9
7	SK-BR-3	3	HER2	20.78	-7.68	205.5
8	HeLa	1	GAPDH	19.12	—	—
9	SK-BR-3	1	GAPDH	19.24	—	—

EXP-2026-0831 RNA Extraction & RT, HeLa & SK-BR-3 Dr. S. Chen Mar 9

EXP-2026-0822 IFN-γ treatment time-course R. Okonkwo Mar 5

SOP-0122 qPCR Setup SOP v3.2 QA Team Feb 28

PRIMER-014 HER2 primers (ERBB2-fwd/rev, IDT lot 20240118) Primer DB Jan 12

PRIMER-002 GAPDH primers (housekeeping ref set) Primer DB Jul 22

2× SYBR Green Master Mix

A25742 · Lot 00845321

In stock

HER2 fwd primer (ERBB2-F)

IDT-014a · Lot 20240118

In stock

HER2 rev primer (ERBB2-R)

IDT-014b · Lot 20240118

In stock

GAPDH primer pair (housekeep)

IDT-002 · Lot 20240118

Low

cDNA stocks (10 µL aliquots)

-80 °C · Freezer B-3 · Shelf 2 · Box B47

×8

Master mix prep (post-aliquot)

-20 °C · Lab fridge · Rack A · Slot 12

×1

Color

Stroke width

Eraser

Use Ctrl + click to add text

{
    "experiment": {
        "id": 314,
        "custom_id": "EXP-2026-0847",
        "title": "qPCR, HER2 Expression in HeLa vs. SK-BR-3",
        "template": "qPCR Experiment",
        "template_version": "v3.2",
        "category": "Molecular Biology",
        "status": "Running",
        "started_on": "2026-05-29",
        "tags": [
            "qpcr",
            "HER2",
            "breast-cancer",
            "IFN-γ",
            "molecular-bio"
        ]
    },
    "main_text": "Objective: Quantify HER2 / ERBB2 mRNA expression in HeLa (low expressor) vs. SK-BR-3 (HER2-amplified) cell lines, with and without 24 h IFN-γ stimulation. GAPDH is used as the endogenous reference for ΔΔCt normalization.\nReagents: 2× SYBR Green Master Mix (Thermo, A25742), HER2 fwd/rev primers (10 µM, IDT lot #20240118), GAPDH fwd/rev primers (10 µM, IDT lot #20240118), cDNA template (50 ng/µL, RT’d from 1 µg total RNA, see RT prep · EXP-2026-0831), nuclease-free water.\nReaction volume: 20 µL per well. Plate: 96-well, technical triplicates, 24 wells total. Instrument: QuantStudio™ 5 (Lab B-204).\nExpected outcome: SK-BR-3 should show ~150-200× higher HER2 expression vs. HeLa baseline. IFN-γ treatment is expected to modestly upregulate HER2 (~3-5×) in both lines.",
    "extra_fields": {
        "Sample & Target": {
            "Cell line": {
                "type": "select",
                "value": "HeLa, SK-BR-3"
            },
            "Treatment": {
                "type": "text",
                "value": "IFN-γ 10 ng/mL, 24 h"
            },
            "Target gene": {
                "type": "text",
                "value": "HER2 (ERBB2)"
            },
            "Reference gene": {
                "type": "text",
                "value": "GAPDH"
            }
        },
        "Reaction Conditions": {
            "Master mix": {
                "type": "select",
                "value": "2× SYBR Green (Thermo A25742)"
            },
            "Reaction volume": {
                "type": "number",
                "value": "20 µL"
            },
            "Primer concentration": {
                "type": "number",
                "value": "0.4 µM"
            },
            "cDNA per well": {
                "type": "number",
                "value": "5 ng"
            }
        },
        "Cycling Program": {
            "Initial denaturation": {
                "type": "text",
                "value": "95 °C × 10:00"
            },
            "Cycles": {
                "type": "number",
                "value": "40"
            },
            "Denature → Anneal": {
                "type": "text",
                "value": "95 °C × 0:15 → 60 °C × 1:00"
            },
            "Melt curve": {
                "type": "check",
                "value": "65 → 95 °C, +0.5 °C/step"
            }
        }
    },
    "attached_files": [
        {
            "filename": "plate_layout.pdf",
            "type": "pdf",
            "size": "142 KB"
        },
        {
            "filename": "quantstudio_results.csv",
            "type": "csv",
            "size": "512 KB"
        },
        {
            "filename": "amp_curves_export.xlsx",
            "type": "xlsx",
            "size": "1.2 MB"
        }
    ],
    "steps": [
        {
            "n": 1,
            "description": "Thaw reagents on ice (SYBR mix, primers, cDNA)",
            "duration": "0:10",
            "complete": true,
            "note": "Recorded at 08:14 by SC"
        },
        {
            "n": 2,
            "description": "Prepare master mix (24 wells × 1.1 excess)",
            "duration": "0:15",
            "complete": true
        },
        {
            "n": 3,
            "description": "Aliquot 18 µL master mix into 96-well plate",
            "duration": "0:08",
            "complete": true
        },
        {
            "n": 4,
            "description": "Add 2 µL cDNA per well (technical triplicates)",
            "duration": "0:12",
            "complete": true
        },
        {
            "n": 5,
            "description": "Seal plate with optical film, vortex, brief spin (1k × 30s)",
            "duration": "0:04",
            "complete": true
        },
        {
            "n": 6,
            "description": "Load plate into QuantStudio 5 (Lab B-204)",
            "duration": "0:02",
            "complete": true
        },
        {
            "n": 7,
            "description": "Run cycling program (validated: 40 cycles + melt)",
            "duration": "1:35",
            "complete": true,
            "note": "Auto-pushed to instrument · run ID 2026-0310-08:22"
        },
        {
            "n": 8,
            "description": "Export raw .eds and CSV to ELabELN",
            "duration": "0:02",
            "complete": true
        },
        {
            "n": 9,
            "description": "Run ΔΔCt analysis (auto-linked GAPDH reference)",
            "duration": "0:01",
            "complete": true
        },
        {
            "n": 10,
            "description": "Apply e-signature for QA sign-off",
            "duration": "0:03",
            "complete": false,
            "note": "Awaiting Dr. Chen"
        }
    ],
    "spreadsheet": [
        [
            "Sample",
            "Replicate",
            "Target",
            "Cq",
            "ΔΔCt",
            "Fold Change"
        ],
        [
            "HeLa",
            "1",
            "HER2",
            "28.42",
            "0.00",
            "1.0"
        ],
        [
            "HeLa",
            "2",
            "HER2",
            "28.51",
            "0.06",
            "0.96"
        ],
        [
            "HeLa",
            "3",
            "HER2",
            "28.39",
            "-0.02",
            "1.02"
        ],
        [
            "SK-BR-3",
            "1",
            "HER2",
            "20.81",
            "-7.65",
            "201.3"
        ],
        [
            "SK-BR-3",
            "2",
            "HER2",
            "20.93",
            "-7.53",
            "184.9"
        ],
        [
            "SK-BR-3",
            "3",
            "HER2",
            "20.78",
            "-7.68",
            "205.5"
        ],
        [
            "HeLa",
            "1",
            "GAPDH",
            "19.12",
            "—",
            "—"
        ],
        [
            "SK-BR-3",
            "1",
            "GAPDH",
            "19.24",
            "—",
            "—"
        ]
    ],
    "links": {
        "experiments": [
            {
                "ref_id": "EXP-2026-0831",
                "title": "RNA Extraction & RT, HeLa & SK-BR-3",
                "owner": "Dr. S. Chen",
                "date": "Mar 9"
            },
            {
                "ref_id": "EXP-2026-0822",
                "title": "IFN-γ treatment time-course",
                "owner": "R. Okonkwo",
                "date": "Mar 5"
            }
        ],
        "resources": [
            {
                "ref_id": "SOP-0122",
                "title": "qPCR Setup SOP v3.2",
                "owner": "QA Team",
                "date": "Feb 28"
            },
            {
                "ref_id": "PRIMER-014",
                "title": "HER2 primers (ERBB2-fwd/rev, IDT lot 20240118)",
                "owner": "Primer DB",
                "date": "Jan 12"
            },
            {
                "ref_id": "PRIMER-002",
                "title": "GAPDH primers (housekeeping ref set)",
                "owner": "Primer DB",
                "date": "Jul 22"
            }
        ]
    },
    "compounds": [
        {
            "name": "2× SYBR Green Master Mix",
            "catalog_lot": "A25742 · Lot 00845321",
            "stock_status": "In stock"
        },
        {
            "name": "HER2 fwd primer (ERBB2-F)",
            "catalog_lot": "IDT-014a · Lot 20240118",
            "stock_status": "In stock"
        },
        {
            "name": "HER2 rev primer (ERBB2-R)",
            "catalog_lot": "IDT-014b · Lot 20240118",
            "stock_status": "In stock"
        },
        {
            "name": "GAPDH primer pair (housekeep)",
            "catalog_lot": "IDT-002 · Lot 20240118",
            "stock_status": "Low"
        }
    ],
    "storage": [
        {
            "name": "cDNA stocks (10 µL aliquots)",
            "location": "-80 °C · Freezer B-3 · Shelf 2 · Box B47",
            "count": "×8"
        },
        {
            "name": "Master mix prep (post-aliquot)",
            "location": "-20 °C · Lab fridge · Rack A · Slot 12",
            "count": "×1"
        }
    ],
    "permissions": {
        "visibility": [],
        "can_write": []
    }
}