Chapter 299 - 203: Professor Sun: If I Can’t Beat Them, Can I Join?

Chapter 299: Chapter 203: Professor Sun: If I Can’t Beat Them, Can I Join?

Hearing this, everyone crowded around.

Wang Xiaoqing also picked up an orange and began to peel it.

Yang Xu casually moved to stand beside Wang Xiaoqing.

He commented offhandedly,

"Looks like you’re on the final wash step for the RNA extraction, right?"

Wang Xiaoqing nodded. "Mhm, it’s the protocol Jiang He just decided on. We’re running this batch with the double chloroform method. Still waiting on the centrifuge results."

"The double chloroform method, that’s a good approach."

Yang Xu continued, "Serum samples aren’t the same as tissue samples. The biggest headache with using Trizol on serum is the excessive protein content. When you’re aspirating the aqueous supernatant, even the slightest slip-up can pull up the intermediate protein layer. That will cause the downstream reverse transcription to fail completely."

"So, it’s absolutely necessary to sacrifice some volume from the first extraction in exchange for purity. You take the supernatant from the first aspiration, add another equal volume of chloroform, vortex vigorously, then centrifuge a second time. That way, the protein contaminants are basically all washed away."

The students all nodded.

Professor Yang really was impressive.

He was saying the exact same thing Jiang He had.

Wang Xiaoqing looked at Yang Xu, a little surprised.

"Director Yang, you’re this well-versed in basic molecular experiments, too?"

Yang Xu feigned modesty. "Medicine is all connected, isn’t it? Clinical and basic research have always complemented each other. Besides, with your protocol, to compensate for the concentration loss from the second extraction, you must have added something before precipitation, right?"

Wang Xiaoqing’s surprise grew. She nodded. "That’s right."

"Five microliters of 5mg/ml glycogen. Glycogen acts as a co-precipitant, doesn’t affect the downstream PCR amplification, and it allows the trace amount of RNA to form a visible pellet at the bottom of the tube. Am I right?"

"Yes, exactly."

Wang Xiaoqing was genuinely impressed now, a hint of admiration coloring her expression.

Yang Xu was inwardly ecstatic, but his expression remained cool and composed.

He turned his head, glanced at Jiang He, and raised his eyebrows.

Jiang He silently peeled an orange, turning his gaze toward the window to look at the sky.

’Go ahead and show off. What can I do? You’re my teacher.’

The break ended.

The experiment resumed.

The next step was the post-precipitation wash with absolute ethanol.

Wang Xiaoqing prepared to use the micropipette herself.

Yang Xu stood beside her, offering soft-spoken reminders under the guise of "guidance."

"Professor Wang, the sample pellet is very small. When you wash it, it’s best to place the tip against the opposite side of the tube wall."

"I know."

"Be slower when you aspirate. Control the rebound of the second stop with your thumb."

"Director Yang, you’re blocking my light."

"Oh, oh, sorry. Habit of mine to focus on the details."

Just then, Professor Sun Changming, who they hadn’t seen in a while, dropped by for a visit.

"Well now, everyone’s hard at work?"

He seemed to be in high spirits today, hands clasped behind his back and looking rather smug.

His gaze swept across the room. Seeing Yang Xu was also there, his smile deepened. "Old Yang is here too? What, the hospital not busy today?"

The smile vanished from Yang Xu’s face. "Here to check on the project’s progress. What are you doing here?"

"Me? I’m here to share some academic experience."

Sun Changming said breezily, "You’re all still working on RNA extraction? Getting used to the equipment?"

Yang Xu replied coolly, "Everything’s fine. Don’t trouble yourself, Professor Sun."

Sun Changming chuckled. "Old Yang, I actually came over today to tell you that at our research institute, we’ve just had a major breakthrough in RNA extraction and purification from digestive tract tumors."

At these words, everyone in the lab looked up.

Sun Changming continued, "As everyone knows, RNA in digestive fluids and serum is extremely prone to degradation. Our team spent a month repeatedly adjusting reagent ratios and finally developed an ultra-purification method."

Wang Xiaoqing said curiously, "Oh? Do tell."

Sun Changming sat up straight and began his long-winded explanation.

"The most important step is abandoning the traditional Trizol method. We adopted a silica-membrane spin-column method combined with multiple enzymatic digestions. First, during the lysis stage, we add a high concentration of proteinase K and digest for an hour in a 56-degree water bath. Then, we pass it through a spin column. To wash away polysaccharide impurities, we specially formulated an elution buffer containing a high concentration of guanidine hydrochloride and isopropanol, and we elute three times."

"Although this process takes more than four hours and is costly, the purity is unquestionable. We’ve consistently achieved an A260/280 ratio of over 1.75! In the current field of liquid biopsy, this is absolutely at the leading edge!"

After speaking, Sun Changming leaned back in his chair, picked up a cup of water from the table, took a sip, and waited for the gasps of amazement.

A purity of 1.75, in ’08, was indeed something to be proud of.

However, everyone in the lab had odd expressions on their faces.

They all looked at each other, seemingly at a loss for words.

Sun Changming frowned. "What? Didn’t you understand? I suppose this enzymatic digestion and column system is a bit complicated. You’ve only just started wet lab work, so take your time..."

"That’s not it, Professor Sun." Chen Hao quietly raised his hand. "I have a question."

"Go ahead," Sun Changming nodded.

Chen Hao asked in a sincere tone, "Since the goal is to solve the problem of protein contamination and degradation, why must you use proteinase K to digest in a water bath for an hour, and also spend so much on spin columns for repeated washes?"

Sun Changming was stunned for a moment. "Without those, how do you remove the protein? How do you purify it?"

Chen Hao replied as if it were the most obvious thing in the world, "Double chloroform extraction with glycogen co-precipitation. It’s the method our boss came up with. You sacrifice a bit of the first supernatant volume, add chloroform and centrifuge again, and all the protein precipitates out. Then you add five microliters of glycogen to get the RNA out. The whole process is done in forty minutes, and the results are great. Why make it as complicated as your way?"

Sun Changming: "?"

After a moment, Sun Changming said in denial, "No, chloroform can indeed remove protein, but if you extract twice, most of the RNA in the aqueous phase will be long gone. Even with glycogen, the concentration definitely won’t meet downstream requirements, let alone the purity. You’d be lucky to get over 1.5."

"But..." Cai Zhuoqun couldn’t help but interject.

"No ’buts.’ Scientific research requires rigor. With a crude method like that, all the data you get will be false positives or straight-up false negatives."

And with impeccable timing...

The new batch from the centrifuge was ready.

Chen Hao said, "Professor Sun, we’ll know if the data is real once we measure it."

Cai Zhuoqun immediately stepped forward, opened the centrifuge lid, added nuclease-free water, and dissolved the pellet.

In front of the NanoDrop spectrophotometer.

One microliter of the sample was pipetted onto the pedestal.

Measure!

The screen refreshed, and a row of data popped up.

Concentration: 14.5 ng/µL.

A260/280: 1.95.

A260/230: 2.03.

Sun Changming: "Huh?"

A ratio between 1.8 and 2.0 is the perfect range for RNA purity.

The 1.75 he was so proud of had taken four hours and all sorts of expensive reagents to achieve.

But this?

What the hell was 1.95!

"Change the tube, measure it again." Sun Changming refused to believe it.

Cai Zhuoqun did as he was told and switched to the second sample.

Concentration: 13.8 ng/µL. A260/280: 1.92.

The third tube.

Concentration: 15.1 ng/µL. A260/280: 1.96.

An incredibly stable high purity, perfect data reproducibility—an absolute, undeniable thrashing.

The smug smile was wiped clean off Sun Changming’s face.

Chen Hao twisted the knife. "Professor Sun, see? Extracted in forty minutes. Aren’t the results much better than yours?"

Yang Xu couldn’t hold it in anymore and burst out laughing. "Old Sun, that method of yours with the one-hour proteinase K water bath, it’s certainly creative. Just a bit time-consuming, labor-intensive, and expensive. No matter, stick to your ideas. I believe in you!"

Sun Changming was silent for a long moment before finally forcing a polite smile.

"Mm... the data is passable, I suppose. However, samples from different tissues have different characteristics. Your method might work on serum, but it may not be applicable to our digestive tract mucus samples. That’s research for you—you have to try many different approaches. Keep it up."

"Oh, I just remembered! I haven’t changed the media for my cells in the incubator yet. I won’t chat with you all any longer today. I’m off."

After saying this, he headed for the door.

Softly he departed, just as softly as he’d come; he flicked his sleeves, not taking a single wisp of cloud with him~

...

「Outside the door.」

Sun Changming clutched his chest, struggling to breathe.

Ugh, this is agonizing!

"Double chloroform... glycogen..."

Leaning on the wall for support as he walked back to his research institute, Sun Changming muttered these words over and over.

’Damn it, how the hell did that kid come up with this? Where did this method even come from? When can I get to do an experiment with him?’

Professor Sun began to consider relying on Jiang He.

’Maybe I should just dissolve my project team. If you can’t beat ’em, join ’em, right?’

’Didn’t Professor Wang already join the student team? Why can’t I?’

’Hmm... is it too late now?’

’Is there an elegant way to surrender? Awaiting replies, it’s urgent.’