Chapter 295: Chapter 200: 35%
The lab conditions at the university weren’t great.
The air conditioning was old, the space was small, and the room was a bit crowded.
The conditions were harsh, but no one felt tired. Everyone was fired up, thinking only of finishing this project as quickly as possible.
The last time Chen Hao had been this motivated was the summer of his sophomore year, when he and Old Jiang went to an internet cafe for a CS 1.6 tournament.
「Times had changed.」
The former M4-addicted teenager had now become a master of the micropipette.
Cai Zhuoqun drew up the nuclease-free water, dispensed it to the bottom of the centrifuge tube, and pipetted it up and down repeatedly.
After observing for a moment, Wang Xiaoqing said, "Let’s test it."
Cai Zhuoqun nodded, ejected the tip, and walked over to the NanoDrop spectrophotometer with the sample.
He used the micropipette to draw 1 microliter of the sample, dropped it onto the measurement pedestal, lowered the arm, and clicked "Measure" on the connected old desktop computer.
Data quickly appeared on the screen.
Lu Xiaolin stopped what he was doing and turned to ask, "What are the numbers?"
Cai Zhuoqun replied, "Concentration is 12 ng/μL, the A260/280 ratio is 1.32, and the A260/230 ratio is 0.7."
Wang Xiaoqing stared at the screen for a while, a bit helpless. "This is already the fourth failed batch..."
The project’s difficulty far exceeded everyone’s expectations—everyone except Jiang He.
Although Jiang He had previously solved the problem of primer dimers during the PCR amplification stage by adding DMSO, solving one problem only led to more...
The current bottleneck was the extraction process.
The concentration of miRNA in blood is extremely low, and it is very easily degraded by RNases in the blood.
The purity wasn’t high enough.
A normal A260/280 ratio should be between 1.8 and 2.0. A value below 1.8 meant the sample had significant protein contamination.
And an A260/230 ratio of only 0.7 indicated a large amount of contamination from phenols or salts.
Using such a sample for the next step, reverse transcription, would be pointless. The impurities would inactivate the reverse transcriptase, and the subsequent PCR amplification would never yield true data.
Cai Zhuoqun tried to find the cause. "Professor Wang, was the amount of chloroform insufficient? When we collected the supernatant, we might have carried over some protein from the interphase."
Wang Xiaoqing shook her head. "For the last batch, we already increased the chloroform ratio by twenty percent and extended the centrifugation time by five minutes. The purity improved slightly, but the concentration dropped below 5 ng/μL."
Lu Xiaolin analyzed from the side, "Serum is full of proteins and has very little free nucleic acid. The Trizol extraction method is inherently flawed for use with serum."
The lab members fell silent.
In later generations, there would be commercial column-based extraction kits designed specifically for serum. They were foolproof and could yield high-purity miRNA in just half an hour.
But in 2008, kits specifically for liquid biopsies were simply not available for purchase in China. Even if they were, the technology was far from mature. They could only use the most basic chemical reagents and painstakingly work through it step by step.
Cai Zhuoqun asked, "Professor Wang, is there any way to fix this?"
Wang Xiaoqing replied, "Don’t ask me. Where’s Jiang He? Where did Jiang He go?"
Chen Hao raised his hand. "Old Jiang said he had to take care of something. He’ll be right back."
Wang Xiaoqing said, "Alright, everyone stop for now. Take a ten-minute break."
’Professor Xiaoqing had learned her lesson.’
’Why struggle with research for half a day when Jiang He could come back and solve it with a single sentence?’
’Better to rest and conserve energy than waste brain cells...’
As everyone went out for a break, Yi Xiangwan and Gu Yizhou went to buy water for everyone.
On the way, Yi Xiangwan, for once, wasn’t joking. He spoke with a hint of worry.
"Yizhou, do you think this project of ours can actually succeed?"
Gu Yizhou glanced at him. "What’s wrong? You want to quit?"
"Hey, no, of course not. It’s just... I was thinking about something. Let’s take a huge step back. Even if we solve the extraction purity problem today, and even if we solve all the subsequent issues with reverse transcription and the PCR system, then what?"
Gu Yizhou frowned.
Yi Xiangwan continued, "How are we supposed to validate it? Since it’s an early screening for pancreatic cancer, in the end, to verify if the model is effective, we’ll need samples from actual patients, right?"
"Moreover, what we need is ’the blood of patients who appear completely healthy but have actually just developed extremely early-stage pancreatic cancer’."
In truth, this was a question that the core team members had all considered.
But everyone was so focused on their work that no one had dared to bring it up.
Because pancreatic cancer is extremely insidious.
By the time patients go to the hospital for abdominal pain or jaundice, it’s often already in the middle to late stages.
In its extremely early stages, pancreatic cancer presents no symptoms in the patient, and it can’t be detected by routine physical exams.
Therefore, the only way to obtain such samples is to rely on a comprehensive serum biobank.
This would mean a hospital storing serum samples from a large population over a long period. Years later, when some of these individuals are diagnosed with pancreatic cancer, researchers could retrieve their serum samples from when they were still healthy to test Jiang He’s early screening model and see if elevated miRNA levels could be detected in their blood from years prior.
But the reality was that in 2008 China, no hospital possessed such a comprehensive, high-quality serum biobank.
Not even Peking Union Medical College Hospital.
"So, if we can’t find the samples for validation, the validation process itself is pointless..."
"What do you want to drink?" Gu Yizhou interrupted him.
"Huh? Oh, uh, Coke is fine. Just a Coke."
Yi Xiangwan took the cold Coke and rolled it against his neck. So cool.
Then he said, "Yizhou, I’m not saying this to discourage the boss. What I mean is..."
"Enough, stop explaining. Why are you overthinking it? Those are problems for the boss to worry about. Our job right now is to get past this extraction step. Don’t let it be that the boss finally manages to get samples from Europe, from Norway, from the United States, and we can’t even extract the RNA. *That* would be embarrassing."
"Damn, you’re right! I really am overthinking things..."
The two of them returned to the lab with the drinks.
They found Jiang He had already returned and was standing in front of the NanoDrop’s screen.
And Professor Wang was obediently reporting the situation:
"We can’t get the purity up. It’s the limitation of Trizol. The protein content in serum is much higher than in tissue, so it’s impossible to avoid carrying over impurities when collecting the supernatant. If we want to avoid impurities, we can only collect a small amount of liquid from the very top layer, but then the RNA concentration won’t meet the requirements for downstream PCR. What do we do?"
Jiang He said calmly, "Didn’t I tell you all before? Add glycogen."
Everyone in the lab froze for a second.
The older members had heard Jiang He mention this before, but none of them knew the specifics of how to do it...
"Oh, my bad. I wasn’t specific enough."
Jiang He then explained, "Before adding the isopropanol to precipitate, add 5 microliters of glycogen at a concentration of 5 mg/mL to the aqueous phase as a co-precipitant. Not only does it act as a carrier to help the trace amounts of RNA aggregate and precipitate, but it also makes the final pellet visible to the naked eye, which makes the subsequent washing steps easier and reduces loss."
Lu Xiaolin thought for a moment and raised a question. "But that doesn’t solve the impurity problem. As long as we use a micropipette to aspirate the aqueous phase, we’ll still get some protein."
Jiang He replied, "That’s why you wash it twice. A double chloroform extraction. After the first centrifugation with chloroform, aspirate the supernatant and transfer it to a new centrifuge tube. Then, add an equal volume of chloroform to this aspirated supernatant, vortex vigorously, and perform a second centrifugation. Simply put, you sacrifice the volume from the first collection to gain purity, and then you bring the concentration back up by adding glycogen as a co-precipitant."
This time, everyone understood.
’Wang Xiaoqing gave herself a mental thumbs-up.’
—’Giving up on thinking was the right choice.’
—’Just listen to Jiang He and everything will be fine!’
—’Tsk, if only every project had a Jiang He. Then I wouldn’t have to pull my hair out in the lab every day.’
Professor Xiaoqing was about to follow in Yang Xu’s footsteps.
It was the same with Yang Xu. Ever since he started collaborating with Jiang He in the operating room, he wanted to bring Jiang He along for every single surgery...
The professors needed to seriously reflect on this behavior of being overly reliant on Jiang He.
The experiment began again under the new protocol.
The high-speed refrigerated centrifuge: lid closed, parameters set: 4 degrees Celsius, 12,000 x g, 15 minutes.
The centrifuge was noisy as it ran.
Jiang He found a random stool, sat down, and continued planning the next stages of the research.
A moment ago, he had stepped out to make a phone call, which was about the serum samples.
"Don’t worry, Jiang He. I’ll go through Europe’s high-spec joint medical research channels and get the serum from the extremely early-stage patients specially approved and air-freighted over on a transnational dry-ice cold chain..."
’Hearing Teacher Gu say that was truly reassuring.’
Everyone else in the lab was a little nervous, all waiting for the results after these 15 minutes.
Yi Xiangwan looked at Jiang He’s back, and a powerful sense of security washed over him.
It was as if, as long as Jiang He was there, all problems were just simple procedural adjustments...
’The boss will definitely be able to handle the serum issue too.’
’I was really worrying too much.’
But what Yi Xiangwan didn’t know was...
...what Jiang He had gone through to gain his current calm.
...
The medical technology of that era was far more advanced than in 2008.
But for Jiang He, it was a meaningless era.
She went quickly. Late-stage pancreatic cancer. From diagnosis to death, it was painful and brief.
In the three years after his wife’s death, he practically lived in the laboratory.
He frantically combed through literature, researching early biomarkers for pancreatic cancer.
’He was always thinking, if it had been discovered just a little earlier, would she have died?’
An almost pathological obsession was the only thing that kept him going.
Back then, Jiang He had also faced this problem with extraction purity.
The kits at that time were effective but extremely expensive.
Moreover, they were still not stable enough for extracting extremely trace amounts of biomarkers.
To find a reliable and cost-effective extraction system, he had fumbled through it alone.
What concentration of glycogen should be added? What would a one-degree difference in the centrifuge’s temperature do? How much shear force damage would an increase of 500 x g in rotational speed cause?
In his past life, he had no one to guide him.
Through countless data errors, tube after tube of failed samples, and night after night of insomnia, he had forged this perfect protocol.
Ten thousand failures in exchange for a single stable reading.
That lab was more spacious than this one, the equipment more advanced, but it was the coldest place he had ever been in his life.
So now, on this fine autumn day, all he had to do was present the correct answer that had been etched into his very bones, like unhesitatingly writing down the unique solution to a math problem.
"BEEP—"
The centrifuge stopped spinning.
Cai Zhuoqun immediately went over and carefully removed the centrifuge tube.
At the bottom of the tube, a white pellet had appeared.
The RNA complex had successfully precipitated after adding glycogen.
He added nuclease-free water to dissolve it.
He drew 1 microliter with the micropipette.
He dropped it onto the NanoDrop pedestal.
Measure!
New data appeared.
Concentration: 14 ng/μL.
A260/280: 1.92.
A260/230: 2.01.
Absolutely perfect data!
Although the concentration was only in the teens, this was already several times higher than with ordinary extraction methods. Most importantly, its purity was extremely high, enough to support downstream reverse transcription PCR.
Wang Xiaoqing was speechless for a long time.
She knew what it meant to achieve this level of purity and concentration for manual serum RNA extraction under the current conditions.
It meant an ultimate mastery of the principles and an understanding of the parameters.
It meant that the few procedural changes Jiang He had casually mentioned had just leapfrogged several years of trial and error in the current industry.
She turned her head and looked at Jiang He.
In this twenty-one-year-old student, she could even feel an awe-inspiring, oppressive sense of scientific authority.
’As an atheist, I now have no choice but to believe the legends about Jiang He being some kind of immortal...’
Chen Hao was the calmest.
He had seen Jiang He’s genius on display more than anyone, so his tolerance was maxed out.
So he said calmly, "Old Jiang, the purity passed."
Jiang He nodded, writing in the lab notebook as he spoke. "Set this as the standard operating procedure for the extraction step. From now on, extract all serum samples using the double chloroform with glycogen protocol."
Jiang He didn’t forget to add a word of praise.
"I’m very satisfied with the progress. Good work, everyone. Keep it up. I have to make a trip to the hospital, so I’ll leave the rest of the work to you."
Everyone replied in unison, "Roger that!"
Jiang He washed his hands, stuffed them in his pockets, and left the lab, looking impossibly cool.
In his mind, there was actually a very clear progress bar.
[miRNA Pancreatic Cancer Early Screening Project Progress: 25%...]
Conquering the extraction process added another ten percentage points.
[miRNA Pancreatic Cancer Early Screening Project Progress: 35%...]
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