Chapter 335 - 228: 100%

Chapter 335: Chapter 228: 100%

"Trizol reagent, ready."

Cai Zhuoqun immediately handed over the reagent bottle.

Jiang He steadily drew a measured amount of Trizol, added it to the serum, and pipetted it up and down repeatedly to lyse the cells.

"Let it sit at room temperature for five minutes." Jiang He glanced at the wall clock.

Five minutes later.

"Chloroform."

He added the chloroform, vortexed it vigorously for fifteen seconds, and let it sit again.

"To the centrifuge."

"Four degrees Celsius, 12,000 g, fifteen minutes."

The centrifuge started.

For those fifteen minutes, no one spoke.

Yi Xiangwan stared at the countdown on the centrifuge and swallowed hard.

Cheng Xiyao and Tang Pei were checking the downstream PCR reaction solutions.

"BEEP—"

The countdown ended.

Jiang He took out the centrifuge tube.

The liquid had clearly separated into three layers.

The top layer was the colorless aqueous phase, containing the RNA they needed.

"Begin the double chloroform extraction."

This was the core technique he had used before to solve the purity problem.

He carefully aspirated the supernatant and transferred it to a new tube.

He sacrificed a portion of the liquid to avoid the protein contamination in the middle layer.

Next, he added an equal volume of chloroform, vortexed it, and centrifuged it again.

When the second centrifugation was finished, Jiang He once again aspirated the supernatant.

"Glycogen."

Jiang He held out his hand.

Wang Xiaoqing immediately handed him the prepared glycogen.

He added 5 microliters of glycogen at a concentration of 5 mg/ml into the aqueous phase as a co-precipitant.

"Isopropanol precipitation. Let it sit at negative twenty degrees for twenty minutes."

Twenty minutes later, they centrifuged it again.

He poured off the supernatant, revealing a tiny white pellet at the bottom of the tube.

"Wash."

He added 75% ethanol, washed it gently, centrifuged, and discarded the liquid.

"Air-dry, then dissolve in nuclease-free water."

The entire extraction process took nearly two hours.

The RNA extraction for the first batch of five samples was complete.

Jiang He took the dissolved sample and walked over to the NanoDrop spectrophotometer.

He pipetted one microliter of the sample and lowered the measurement arm.

Everyone gathered around.

Data appeared on the screen:

Concentration: 15 ng/μL.

A260/280: 1.95.

A260/230: 2.03.

Extremely pure data.

Despite the international shipping and repeated freeze-thaw cycles, they had still managed to obtain perfect samples with this extraction method.

"Extraction successful," Jiang He said calmly. "Process the rest of the samples using this standard. Professor Wang, prepare for reverse transcription."

"Understood." Wang Xiaoqing immediately took the sample and moved to another workstation.

The reverse transcription process was relatively automated.

Using specific stem-loop primers, the extremely minute amount of miRNA was reverse transcribed into cDNA.

Time ticked by, second by second.

Outside the window, the sky gradually darkened.

No one suggested eating.

Everyone worked like a relay team, completing each step in succession.

At 8:00 p.m.

The cDNA for all fifty samples was ready.

The most critical step had arrived.

"Prepare to load the qPCR machine," Jiang He said.

Yi Xiangwan and Gu Yizhou carefully added the prepared PCR reaction system into the 96-well plate.

Each well contained primers, fluorescent dye, a cDNA template, and the enzyme.

This was the system they had previously optimized under the extremely low concentration of a 10,000-fold dilution.

Jiang He picked up the 96-well plate and placed it in the reaction block of the qPCR instrument.

He closed the lid.

Entered the parameters.

Set the cycling program.

"Begin run."

The machine started.

The detection process would take two hours.

To everyone in the lab, those two hours felt as long as a century.

If the amplification curve was a smooth, flat line, it would mean the patients’ blood from two or three years ago had no abnormal expression of these specific miRNAs.

That would mean the early screening model had failed.

If the curve rose beautifully and the Ct value (cycle threshold) was within the valid range, it would prove the model worked.

Jiang He pulled over a chair and sat in front of the computer.

He didn’t speak, and no one else dared to either.

Yi Xiangwan paced back and forth by the workstation.

"Stop pacing," Gu Yizhou said, annoyed.

"I’m nervous, Senior Brother! What if... I mean, what if the serum from Europe degraded? Or what if the biomarkers we’re looking for aren’t actually released in the very early stages?"

"Shut up," everyone interrupted him in unison.

Yi Xiangwan quickly raised his hands. "Sorry!"

The first stage of denaturation, annealing, and extension cycles ended.

The screen was still blank.

"Why aren’t there any curves yet?" Lu Xiaolin’s palms were already sweating.

Cai Zhuoqun said in a low voice, "What’s the rush? These are extremely early-stage samples. The concentration is low, so the Ct value is bound to be high."

The thirtieth cycle.

Still no significant rise.

The thirty-second cycle.

Suddenly.

On the screen’s coordinate axis, the tail of the first curve showed an upward angle.

Everyone held their breath.

The thirty-third cycle.

The upward angle began to steepen.

Immediately after, the second, third, and fifth curves...

The thirty-fifth cycle.

The screen erupted with dense amplification curves!

One after another, smooth logarithmic amplification curves shot up from the baseline!

There was no messy, non-specific amplification whatsoever!

And on the other side, the melt curve interface showed beautiful, uniform single peaks!

"HOLY SHIT..."

Yi Xiangwan blurted out a curse.

Wang Xiaoqing’s breathing grew ragged. "The internal control curve is stable, no pipetting error... It’s here, Jiang He! Compared to the 300 healthy baselines we established, all fifty samples show significant overexpression of the characteristic miRNAs!"

Chen Hao gasped.

Lu Xiaolin and Cai Zhuoqun exchanged a glance.

Fifty serum samples from extremely early-stage pancreatic cancer—fifty seeds of disease, hidden beneath the veneer of health.

This meant that in 2008, they had managed to snatch the king of cancers from the bloodstream.

This achievement could advance the detection of cancer by a full two to three years!

Jiang He remained seated, a progress bar ticking in his mind.

[miRNA Pancreatic Cancer Early Screening Project Progress: 100%]

He had completed this step.

This single step accounted for the remaining fifty percent of the project.

In his past life, he had pored over countless papers, spent endless nights in the lab, an obsession born from ten thousand failures just to get one stable reading.

In this life, he had started from scratch, accumulating funds and recruiting a team step by step...

And now, all of it had finally borne fruit.

He closed his eyes.

Shen Yu’s face flashed before him.

"Dr. Jiang, you’re back. There’s something I want to tell you..."

Jiang He gritted his teeth.

’This time, I won’t let you say those words. I swear it.’

"Boss!!!"

Yi Xiangwan rushed over and hugged Jiang He from behind. "We did it!! HOLY SHIT! FUCKING AWESOME! Boss, we really did it!"

Gu Yizhou rushed over too, roaring, "FUCKING AWESOME! FUCKING AWESOME!"

Everyone surged forward, crowding around them.

"We did it!"

"The data is perfect!!!"

"We nailed it! We nailed it!"

"Boss, you’re a god! This system is incredible!"

Tears streamed down the faces of Cheng Xiyao and Tang Pei.

Even the usually composed Professor Wang Xiaoqing was too excited to know what to say.

The lab erupted in celebration.

Everyone celebrated like mad, hugging and slapping each other on the back.

Jiang He was shaken so much he could barely stay in his seat.

He let them vent their emotions, a smile finally gracing his lips.

He raised a hand and pressed it downwards.

The lab quieted down a little, and everyone stared at him with burning intensity.

Jiang He straightened his white lab coat and said in a calm voice:

"Seal the data and prepare the report."

"Brothers, it’s time we all took a trip to Baltimore."